Method for preparing substituted amino alcohol compounds

ABSTRACT

Disclosed is a process for preparing compounds having a straight or branched aliphatic hydrocarbon structure of formula I: ##STR1## In formula I, n is an integer from one to four and m is an integer from four to twenty. Independently, R 1  and R 2  are hydrogen, a straight or branched chain alkyl, alkenyl or alkynyl of up to twenty carbon atoms in length or --(CH 2 ) w  R 5 . If R 1  or R 2  is --(CH 2 ) w  R 5 , w may be an integer from one to twenty and R 5  may be an hydroxyl, halo, C 1-8  alkoxyl group or a substituted or unsubstituted carbocycle or heterocycle. Alternatively, R 1  and R 2  may jointly form a substituted or unsubstituted, saturated or unsaturated heterocycle having from four to eight carbon atoms, N being a hetero atom of the resulting heterocyle. R 3  may be either hydrogen or C 1-3 . In the compounds, a total sum of carbon atoms comprising R 1  or R 2 , (CH 2 ) n  and (CH 2 ) m  does not exceed forty. R 4  is a terminal moiety comprising a substituted or unsubstituted, oxidized or reduced ring system, the ring system having a single ring or two to three fused rings, a ring comprising from three to seven ring atoms. The disclosed compounds are effective agents to inhibit undesirable responses to cell stimuli.

This is a Division of U.S. application Ser. No. 08/303,842, filed Sep.8, 1994, which is a Continuation-in-Part of application Ser. Nos.08/152,650, filed Nov. 12, 1993 and 08/164,081, filed Dec. 8, 1993,which are Continuation-in-Part Applications of application Ser. No.08/040,820, filed Mar. 31, 1993, now abandoned.

FIELD OF THE INVENTION

The invention provides a group of compounds that are effective agents toinhibit specific cellular signaling events often induced by inflammatorystimuli, to act as anti-inflammatory or immunosuppressive agents, to actas cytotoxic agents for treatment of cancers, or to be directly orindirectly antimicrobial to yeast or fungal infections. Morespecifically, the inventive compounds have at least one amino alcohol(or derivative thereof) functional group attached to a terminal moietyvia an aliphatic hydrocarbon.

BACKGROUND OF THE INVENTION

Pentoxifylline 1-(5-oxohexyl)-3,7-dimethylxanthine!, abbreviated PTX, iswidely used medically for increasing blood flow. U.S. Pat. Nos.3,422,107 and 3,737,433, both to Mohler et al., disclose PTX.Metabolites of PTX were summarized in Davis et al., "Microbial Models ofMammalian Metabolism: Microbial Reduction and oxidation ofPentoxifylline," Applied and Environmental Microbiology, Vol. 48, No. 2,pages 327-381, August 1984, and Bryce et al., "Metabolism andPharmacokinetics of ¹⁴ C-Pentoxifylline in Healthy Voluteers,"Arzneim.-Forsch./Drug Res. Vol. 39, No. 4, pages 512-517, 1989. Ametabolite of PTX is 1-(5-hydroxyhexyl)-3,7-dimethylxanthine, designatedM1. M1 was also disclosed as increasing cerebral blood flow in U.S. Pat.Nos. 4,515,795 and 4,576,947 to Hinze et al. Other metabolites include1-(5-pentoyl)-3,7-dimethylxanthine carboxylic acid, designated M4, and1-(4-butyl)-3,7-dimethylxanthine carboxylic acid, designated M5. Inaddition, U.S. Pat. Nos. 4,833,146 and 5,039,666 to Gebert et al. andNovick, respectively, disclose use of tertiary alcohol analogs ofxanthine-containing compounds for enhancing cerebral blood flow.

PTX and its known metabolites thereof have been shown to have in vivoactivity in specific biologic systems. U.S. Pat. No. 4,636,507 toKreutzer et al. describes an ability of PTX and M1 to enhance chemotaxisin polymorphonuclear leukocytes responding to chemotaxis stimulation. Inaddition, PTX and related tertiary alcohol substituted xanthines inhibitactivity of certain cytokines to affect chemotaxis as described in U.S.Pat. Nos. 4,965,271 and 5,096,906 to Mandell et al. Furthermore, byco-administering PTX and GM-CSF, patients undergoing allogeneic bonemarrow transplant exhibited decreased levels of tumor necrosis factor,TNFα. Bianco et al., "Pentoxifylline (PTX) and GM-CSF Decrease TumorNecrosis Factor (TNFα) Levels in patients undergoing allogeneic BoneMarrow Transplantation (MBT)," Blood, Vol. 76, No. 1, Suppl. 1 (522),page 133a, 1990. Reduction in assayable levels of TNFα was accompaniedby reduced BMT-related complications. However, in normal volunteers,TNFα levels were higher among PTX recipients. Therefore, elevated levelsof TNFα are not the primary cause of such complications.

Further research with PTX, its metabolites and their activity relatingto various biologic systems spurred investigations with potentialtherapeutic agents heretofore unknown. These agents were identified aspotential therapies for treating or preventing disease by inhibitingsecondary cellular response to an external or in situ primary stimuli.These investigations sought efficacious, therapeutic compounds, whichwould be safe and effective for human or animal administration and wouldmaintain cellular homeostasis in the presence of a variety ofdeleterious stimuli.

Many diseases are difficult to treat because they have complexmechanisms of action, and multiple, adverse effects on a subject. As anexample, cancer has been difficult to treat for this and other reasons.Precise causes of cancer remain unknown. Malignant tumor growth resultsfrom many physiologic factors. Cancer cells metastasize (i.e., breakthrough blood vessels and travel to distant body sites) and secreteenzymes called metalloproteases, which "break down" blood vessel walls,allowing the cancer cells to enter the bloodstream and form remotetumors (proteolysis). In addition, tumor cell adhesion receptors(integrins) effect attachment--necessary for tumor residence inorgans--of tumor cells to blood vessel walls and normal organs. Cancercells also secrete certain proteins, such as bFGF, that stimulate newblood vessel development (angiogenesis), these new blood vesselssupplying nutrients to promote malignant tumor growth.

Conventional antineoplastic therapies, such as, for example,antimetabolites, alkylating agents and antitumor agents (which target orinterfere with DNA and/or synthesis of DNA or its precusors), andbiologic therapies (including selective interferons, interleukins andother factors) have significant adverse side effects in patients, notlimited to acute toxicity due to effects on rapid-proliferating tissues,such as bone marrow and oral epithelium, myelosuppression and mucositis,renal failure and neurological, hepatic or pulmonary toxicity. Thus, forexample, a cancer therapy which effectively prevented, reduced oreliminated malignant tumors without causing deleterious side effectswould provide previously unknown treatment.

Compounds disclosed herein and discovered in search of potential diseasetreatments which would prevent or treat a disease with minimal or noadverse side effects, have biologic activity in multifarious, predictiveassays. The inventive compounds exhibit utility in preventing anundesireable cellular response to noxious stimuli. Results frompredicitive assays indicate that these inventive compounds havepotential as therapies in treating a broad spectrum of clinicalindications, acting via a variety of disease mechanisms. However, allthese mechanisms appear to affect the second messenger pathway. Resultsof this research are the subject matter of this disclosure, thecompounds discussed herein having novel structures and remarkable andsurprising properties heretofore unknown.

SUMMARY OF THE INVENTION

The invention provides compounds useful in a large variety oftherapeutic indications for modulating disease by intracellularsignaling through specific intracellular signaling pathways. Inaddition, the compounds and pharmaceutical compositions are suitable fornormal routes of therapeutic administration (e.g., parenteral, oral,topical, etc.) for providing effective dosages of a therapeuticcompound. The compounds include resolved enantiomers and/ordiastereomers, hydrates, salts, solvates and mixtures thereof, thecompounds having a straight or branched aliphatic hydrocarbon structureof formula I: ##STR2##

In formula I, n is an integer from one to four and m is an integer fromfour to twenty. Independently, R₁ and R₂ are hydrogen, a straight orbranched chain alkyl, alkenyl or alkynyl of up to twenty carbon atoms inlength or --(CH₂)_(w) R₅. If R₁ or R₂ is --(CH₂)_(w) R₅, w may be aninteger from one to twenty and R₅ may be an hydroxyl, halo, C₁₋₈ alkoxylgroup or a substituted or unsubstituted carbocycle or heterocycle.Alternatively, R₁ and R₂ may jointly form a substituted orunsubstituted, saturated or unsaturated heterocycle having from four toeight carbon atoms, N being a hetero atom of the resulting heterocyle.R₃ may be either hydrogen or C₁₋₃. Preferred compounds may have one ofR₁ or R₂ and R₃ that form a substituted or unsubstituted linking carbonchain, having from one to four carbon atoms. This R₁ /R₃ or R₂ /R₃linking chain will join the O and N in a cyclic structure, an integersum equal to n+a number of carbon atoms in the linking carbon chainbeing less than six.

In the compounds, a total sum of carbon atoms comprising R₁ or R₂,(CH₂)_(n) and (CH₂)_(m) does not exceed forty. R₄ is a terminal moietycomprising a substituted or unsubstituted, oxidized or reduced ringsystem, the ring system having a single ring or two to three fusedrings, a ring comprising from three to seven ring atoms. However, if R₄is phthalimide, m of formula I is not less than five.

The compounds may include resolved enantiomers and/or diastereomers,hydrates, salts, solvates and mixtures thereof that have a straight orbranched aliphatic hydrocarbon structure of formula II: ##STR3##

In the above formula II, n, m, R₃, and R₄ are defined as provided informula I above. R₆ and R₇ are hydrogen, a straight or branched chainalkane, alkene or alkyne of up to twenty carbon atoms in length, or--(CH₂)_(x) R₈, at least one of R₆ or R₇ being --(CH₂)_(x) R₈. Informula II, x is an integer from zero to fourteen and R₈ is a moietyhaving a general structure as provided in formula III ##STR4##

In formula III above, m, R₃, and R₄ are defined as provided in formula Iabove. Z is N or CH and p is an integer from zero to four. R₉ is H or astraight or branched chain alkane, alkene or alkyne of up to twentycarbon atoms in length.

The invention provides a pharmaceutical composition comprising ancompound or a pharmaceutical salt, hydrate or solvate thereof and apharmaceutically acceptable excipient. The pharmaceutical compositionmay be formulated for oral, parenteral or topical administration to apatient.

The invention includes a method for treating an individual having avariety of diseases. The disease is characterized by or can be treatedby inhibiting an immune response or a cellular response to external orin situ primary stimuli, the cellular response being mediated through aspecific phospholipid-based second messenger acting adjacent to a cellmembrane inner leaflet. The second messenger pathway is activated inresponse to various noxious or proliferative stimuli characteristic of avariety of disease states. Biochemistry of this second messenger pathwayis described herein. More specifically, the invention includes methodsfor treating or preventing clinical symptoms of various disease statesor reducing toxicity of other treatments by inhibiting cellularsignaling through a second messenger pathway involving signaling throughphosphatidic acid and through glycan phosphatidylinostinol (Gly PI).

The compounds are of particular significance for inhibiting IL-2-inducedproliferative response. IL-2 signaling inhibition is potentially usefulin the treatment of numerous disease states involving T-cell activationand hyperproliferation. Exemplary autoimmune diseases treated byinhibiting IL-2 signaling are lupus, scleroderma, rheumatoid arthritis,multiple sclerosis, glomerula nephritis as well as potentialmalignancies, including but not limited to, chronic myelogenous leukemiaas well as others.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are dose response curves for both cyclosporin A (CsA,FIG. 1A) and various compounds (FIG. 1B) for murine thymocyteproliferation co-stimulated by Concanavalin A (ConA) and interleukin-2alpha (IL-2).

FIGS. 2A and 2B illustrate immune modulating activity of compounds nos.5 and 26 (see corresponding names and structures below) in an assaydetermining proliferative PMBC response to allogeneic stimulation usinga two-way, mixed lymphocyte reaction.

FIG. 2C and 2D report activity data for compounds nos. 27 and 13 andIC₅₀ data, respectively in a mixed lymphocyte reaction.

FIG. 2E shows percent viability for five compounds (correspondingchemical names below) in a mixed lymphocyte assay measuring immunesuppression activity of the compounds tested.

FIG. 2F shows percent viability results from a mixed lymphocyte reactionassay.

FIG. 3A reports results obtained for several compounds exhibitinginhibitive effects on murine thymocyte proliferation.

FIG. 3B shows comparative results from inhibition assays forco-stimulated thymocyte proliferation.

FIG. 4 reports IC₅₀ values for compounds nos. 27 and 28 in a murinesplenocyte proliferation (anti-mu stimulated assay).

FIGS. 5A and 5B are frequency histograms of measurements for 20,000cells in an assay illustrating IL-2 (alpha chain CD25) receptorexpression.

FIG. 6 shows results for compounds nos. 49 and 50 as compared with CsAin an inhibitive assay for production of IL-2 in murine thymocytes.

FIGS. 7 and 8 report dose response results from an in vitro assayemphasizing cellular or T-cell immune response.

FIGS. 9A and 9B report inhibitive activity for the compounds, ascompared with CsA on direct IL-2-induced proliferation in a murinecytotoxic T-cell line, CT6.

FIG. 10 illustrates activity of the compounds in anti-yeast andanti-fungal assays.

FIG. 11 reports activity data for compounds in an assay designed todetect potential antigen specific anergy-induction.

FIG. 12 shows activity results for compounds nos. 28, 30, 31, 33 and 29in an assay useful in predicting therapeutic activity for preventing ortreating restenosis, atherosclerosis and coronary artery disease.

FIG. 13 shows cytotoxic results for compounds in a human stromalcell/PDGF stimulation assay.

FIG. 14 reports inhibitive activity results for compounds nos. 27, 28,29, 30, 31, 32 and 34 in an assay measuring inhibitive effects in aPDGF/IL-1β co-stimulation.

FIG. 15 illustrates a dose response curve for selected compounds in anassay measuring PDGF-induced proliferation of Balb/3T3 cells.

FIG. 16 reports suppressive results for selected compounds in a murinethymocyte ConA/IL-2 co-stimulation assay.

FIG. 17 compares IC₅₀ and ID₅₀ data for several compounds.

FIG. 18 reports cytotoxicity results for compound no. 27 for transformed(Ras 3T3) cells and non-transformed (normal) cells.

FIG. 19 shows data demonstrating inhibitory effects of compound no. 58on PDGF-induced proliferation of human aortic smooth muscle cells(aortic SMC).

FIGS. 20A and 20B show the effects of compound no. 58 on aFGF and bFGF-induced proliferation in human aortic smooth muscle cells (aortic SMC).

FIGS. 21A and 21B show inhibitory activity of compound no. 35 and CsA,respectively, on murine thymocytes, co-stimulated with ConA and IL-2.

FIGS. 21C and 21D illustrate inhibitory effects of compound no. 35 andCsA, respectively, on IL-2-induced proliferation of cytotoxic CT-6cells.

FIG. 22 reports activity results for compound no. 58 on vascularendothelial growth factor (VEGF)-induced proliferation in a humanumbilical vein endothelial cell line (HUVEC).

FIG. 23 is a series of frequency histograms obtained from flowcytometric analysis of HUVEC cells.

FIG. 24 illustrates inhibitive results obtained for compounds nos. 50and 57 on THP-1 cell adhesion to IL-1β-activated HUVEC.

FIGS. 25A and 25B show the effects of compound no. 58 on aFGF andbFGF-induced proliferation in pulmonary smooth muscle cell.

FIGS. 26A and 26B illustrate inhibition of VCAM-1 or ICAM-1 expression,respectively, in HUVEC activated by TNFα by compound no. 58.

FIG. 27 illustrates that compound no. 58 inhibits TNFα release bycompound no. 58 using a human whole blood ex vivo assay.

FIG. 28 reports results for compound no. 50 in the PDGF-inducedproliferation of Balb/3T3 cells.

FIG. 29 shows that TNFα substantially increased expression of the 92 kDmatrix metalloprotease (MMP) and moderately increased production of the72 kD MMP and that the presence of compound no. 50 blocked theTNFα-stimulated expression of each MMP.

FIG. 30 shows anti-proliferative activity and FIG. 31 reportsanti-clonogenicity activity of compound no. 50 with HT-29 cells.

FIGS. 32 and 33 illustrate cytotoxicity and concentration dependence ofcompound no. 50 against 3LL cells (Lewis lung carcinoma).

FIG. 34 shows that compound no. 50 lacks cytotoxic activity in normalhuman bone marrow stromal cells.

FIGS. 35 and 36 show the effects of compound no. 50 on matrigel invasionand viability in 3LL cells.

FIG. 37 illustrates VEGF-induced proliferation of HUVEC as a predictiveadhesion assay.

FIG. 38 shows the effect of compound no. 50 on THP-1 adherence toIL-1β-stimulated HUVEC.

FIG. 39 illustrates the effect of compound no. 50 on VCAM-1 surfaceexpression of TNFα-stimulated HUVEC.

FIG. 40 illustrates the effect of compound no. 50 on ICAM-1 surfaceexpression of TNFα-stimulated HUVEC.

FIGS. 41A, 41B and 41C illustrate the results from an in vivo study inmice with B16 melanoma cells.

FIGS. 42A and 42B illustrate T- and B-cell response assays of micetreated with compound no. 50 in a B16 melanoma cell assay.

FIG. 43 reports results in an in vivo experiment showing that compoundno. 50 can arrest growth of 3LL cells in mice.

FIG. 44A and 44B illustrate platelet and neutrophil counts,respectively, of sacrificed mice in a Lewis Lung carcinoma assay.

FIGS. 45A, 45B and 45C illustrate a photographic comparison of lungsfrom 3LL exposed mice with or without treatment using compound no. 50.

FIG. 46 reports results obtained in an assay investigating compoundno.'s 45 effect on IL-2-mediated proliferation

FIG. 47 illustrates that compound no. 45 inhibits CT-6.1 proliferationas measured by cell number.

FIGS. 48A, 48B, and 48C illustrate that compound no. 45 also inhibitsmitogenic responses to IL-2, IL-4 and IL-7.

FIG. 49 shows that compound no. 45 also inhibited murine thymocyteproliferation in response to ConA and IL-2.

FIGS. 50A and 50B illustrate that compound no. 45 inhibits a murinemixed tumor lymophocyte culture (MTLC) and a human mixed leukocytereaction (MLR), respectively.

FIG. 51 illustrates an effect of the delayed addition of compound no. 45on co-stimulated thymocyte proliferation.

FIG. 52 illustrates that compound no. 45 inhibits anti-CD3 stimulatedsplenocyte proliferation.

FIGS. 53A and 53B illustrate that compound no. 45 does not inhibitT-cell receptor (CD3) mediated signaling.

FIGS. 54A and 54B illustrate that compound no. 45 does not inhibitanti-CD3 mediated upregulation of the IL-2 receptor alpha subunit.

FIG. 55 illustrates that compound no. 45 does not inhibit IL-2 receptorbeta (p70) subunit internalization.

FIGS. 56A and 56B illustrate that the compound no. 45 also inducesantigen specific T-cell anergy.

FIG. 57 illustrates that compound no. 45 inhibits B cell proliferation.FIGS. 58A and 58B illustrate that compound no. 45 does not inhibitCD28-mediated IL-2 release.

FIGS. 58C, 58D and 58E illustrate that compound no. 45 inhibits IFN-γrelease by blocking IL-2 signaling.

FIGS. 59A, 59B and 59C report assay results investigating the effect ofcompound no. 45 on cytokine release from anti-CD3-stimulated mousesplenocytes.

FIG. 60A shows inhibition of proliferation in an MTLC assay withcompound no. 45.

FIG. 60B and 60C illustrate that compound no. 45 does not inhibit eitherIL-2 or TNFα release from the MTLC.

FIG. 60D shows that compound no. 45 inhibits IFN-γ release.

FIG. 61 illustrates that compound no. 45 does not have an effect onprostaglandin E2 release from IL-1α-stimulated human foreskinfibroblasts, HS68.

FIGS. 62A and 62B report inhibition of THP-1 adhesion to TNFα orIL-1β-stimulated HUVEC.

FIGS. 63A and 63B illustrate that compound no. 45 inhibits adhesionreceptor expression on HUVEC.

FIG. 64 reports that compound no. 45 inhibits PDGF-BB-induced murineBALB/3T3 proliferation.

FIGS. 65A-65F illustrate inhibition of proliferation in either humanaortic or pulmonary smooth muscle cells (SMC) by compound no. 58.

FIGS. 66A and 66B are a dose response and cytoxicity curves,respectively, for inhibition of proliferation in Balb/3T3 cells bycompound no. 58.

FIGS. 67A and 67B illustrate that compound no. 58 inhibits VEGF-inducedproliferation in HUVEC and EGF-induced proliferation in Swiss/3T3 cells,respectively.

FIG. 68 illustrates an effect of the delayed addition of compound no. 58to Balb/3T3 proliferation in response to PDGF-BB.

FIG. 69 illustrates that compound no. 58 inhibits PDGF- inducedproliferation in Balb/3T3 cells to a greater extent than serum-inducedproliferation,

FIG. 70 illustrates that endothelial cell migration is inhibited bycompound no. 58.

FIG. 71 illustrates that compound no. 58 does not inhibit chemotaxis ofhuman smooth muscle cells (SMC) to PDGF.

FIGS. 72A and 72B illustrate that compound no. 58 inhibits THP-1 celladhesion to either TNFα or IL-1β-stimulated HUVEC (respectively).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a genus of compounds which can control cellularbehavior by a particular phase of a secondary messenger pathway system(Bursten et al., "Interleukin-1 Rapidly Stimulates LysophosphatidateAcyltransferase and Phosphatidate Phosphohydrolase Activities in HumanMesangial Cells," J. Biol. Chem., Vol. 266, No. 31, pages 20732-20743,Nov. 5, 1991). The second messengers are lipids or phospholipids and usethe following abbreviations:

PE=phosphatidyl ethanolamine

LPE=lysophosphoethanolamine

PA=phosphatidic acid

LPA=lysophosphatidic acid

DAG=diacylglycerol

LPLD=lysophospholipase-D

LPAAT=lysophosphatidic acid acyl transferase

PAPH=phosphatidic acid phosphohydrolase

PLA2=phospholipase A2

PLD=phospholipase D

PAA=phosphoarachidonic acid

PC=phosphatidyl choline

PIG-PLD=Glycanphosphotidylinositol phospholipase D

Gly PI=glycanphosphotidylinositol

Gly I=glycan inositol

"remodeled" PA, cyclic pathway=PAA, LPA, PA and DAG intermediatessubstituted with 1-saturated, 2-linoleoyl or 1,2-dioleoyl,dioleoy/1,2-sn-dilinoleoyl at the indicated sn-1 and sn-2 positions.

"Classical PI Pathway"=PI, DAG, PA intermediates substituted with1-stearoyl, 2-arachidonoyl fatty acyl side chains.

"PLD-generated PA"=PE, PC, LPA, PA and DAG intermediates substitutedwith, e.g., 1,2-sn-dioleoyl-, 1-alkyl, 2-linoleoyl-, and 1-alkyl,2-docosahexaenoyl-side chains.

Lysophosphatidic acid transferase (LPAAT) effects the synthesis ofphosphatidic acid (PA) from lysophosphatidic acid (LPA) by incorporationof an acyl group from acyl CoA. Hydrolysis of the phosphate moiety by PAphosphohydrolase (PAPH) results in the formation of DAG. These aspectsof the pathway appear to be activated immediately (within a minute) uponstimulation by a primary stimulus (e.g., a cytokine such as IL-1β, IL-2or TNFα) acting at a receptor on a cellular surface. An immediatedetectable effect is an elevation of levels of PA and DAG.Administration of the compounds of the invention reverse this elevation.

The compounds and pharmaceutical compositions of the invention includeinhibitors of subspecies of LPAAT and PAPH enzymes with substratespecificity for intermediates with 1,2-diunsaturated and 1-alkyl,2-unsaturated subspecies. Each membrane phospholipid subclass (e.g., PA,PI, PE, PC and PS) reaches a-stable content of characteristic fatty acylside chains due to cyclic remodeling of the plasma membrane as well asturnover for each subclass. PA is often stable, but present inrelatively small quantities. PA in resting cells consists mostly ofsaturated acyl chains, usually consisting of myristate, stearate andpalmitate. In resting cells, PC's acyl side chains consist mostly ofacyl palmitate in the sn-1 position and oleate in the sn-2 position. PEand PI are predominantly composed of sn-1 stearate and sn-2arachidonate.

Due to this characteristic content of acyl groups in the sn-1 and sn-2positions, the origin of any PA species may be deduced from the chemicalnature of its acyl groups in the sn-1 and sn-2 positions. For example,if PA is derived from PC through action of the enzyme PLD, the PA willcontain the characteristic acyl side chains of PC substrate passedthrough the second messenger pathway. Further, the origin of any 1,2sn-substrate species may be differentiated as to its origin. However, itis important to know whether or not each phospholipid species passesthrough a PA form previous to hydrolysis to DAG. The lyso-PA that isconverted to PA and then to DAG may be shown. The complexities of thissecond messenger pathway can be sorted by suitable analyses by fattyacyl side chain chemistry (i.e., by thin layer chromatography,gas-liquid chromatography, or high pressure liquid chromatography) ofintermediates in cells at various time points after stimulation of thesecond messenger pathway.

In certain meseachymal cells, such as neutrophils and rat or humanmesangial cells, several signaling pathways may be activated in tandem,simultaneously or both. For example, in neutrophils, F-Met-Leu-Phestimulates formation of PA through the action of PLD, followed in timeby formation of DAG through the action of PAPH. Several minutes later,DAG is generated from PI through the classical phosphoinositide pathway.In many cells, DAG is derived from both PA that is being remodeledthrough a cycle whereby PA is sn-2 hydrolyzed by PLA2, followed by sn-2transacylation by LPAAT, and a PLD-pathway from PA that is generatedfrom either PE or PC or both substrates by PLD.

The present second messenger pathway involves substrates withunsaturated fatty acids in the sn-2 position other than arachidonate andthose sub species of PAPH and LPAAT that are not involved in normalcellular housekeeping functions that are part of the classical PIpathway.

The PAPH and LPAAT enzymes involved in the present second messengerpathway are exquisitely stereo specific for different acyl side chainsand isomeric forms of substrates. Therefore, the compounds arepreferably, substantially enantiomerically pure if a chiral center ispresent.

An additional signaling pathway associated with inflammatorytransduction and cell membrane perturbation generates a separate PAspecies, enriched in myristate and derived from GlyPI, as describedabove. Under these signaling conditions, the compounds preventactivation of or directly inhibit the PiG-PLD, hydrolyzing GlyPI to PAand GlyI. In some tumor cells and TNFα-activated cells (i.e., Type IIreceptors), the compounds' efficacy may be dual inhibition of both LPAATand GlyPI hydrolysis. Experimental results confirm this inhibitiveeffect. Stimulation of CT-6 cells with IL-2 results in rapid hydrolysisof GlyPI species 15-45 seconds after stimulation, followed by rapidresynthesis of GlyPI. The compounds prevent this hydrolysis andstimulate GlyPI synthesis, resulting in a significant GlyPI increasethroughout stimulation without evidence of hydrolysis or formation ofGlyPI-derived PA. Stimulation of human umbilical vein endothelial cellswith TNFα results in LPAAT-derived and Gly-PI-derived PA species. Thecompounds inhibit formation of both PA species. Accumulation of lyso-PAand Gly-PI results.

Therapeutic Uses of the Inventive Compounds

The specific activation or inhibition of the second messenger cellsignaling pathway, as described above, activated primarily by variousstimuli, suggests that the inventive compounds are useful in treating awide variety of clinical indications. Moreover, in vitro and in vivodata, presented herein, provides predictive data that a wide variety ofclinical indications, having similar effects on the specific secondmessenger pathway, may be treated by the inventive compounds, whichspecifically inhibit the second messenger pathway mediated through, forexample, inflammatory cytokines. In fact, the mechanism of action forthe compounds explains why these compounds have multifarious clinicalindications.

Activation of the second messenger pathway is a major mediator ofresponse to stimuli and results in intracellular signalling that leadsto clinical manifestations of, for example, acute and chronicinflammation, autoimmune diseases and cancer cell growth. However, allinhibitors (i.e., the inventive compounds) do not inhibit all enzymes ofthis second messenger pathway. Signals mediated by the present secondmessenger pathway include, for example, those cellular responses of LPSdirectly, T- and B-cell activation by antigen, cellular responses tofirst messengers (e.g. inflammatory cytokines), such as, IL-1βand TNFα,growth stimulated by transformations including, but not limited to,activated oncogenes (e.g., ras, abl, her2-neu and the like), smoothmuscle cell proliferation stimulated by platelet derived growth factor(PDGF), b-FGF, epidermal growth factor (EGF), vascular endothelialgrowth factor (VEGF) and IL-1β, T- and B-cell growth stimulation byIL-2, IL-4 or IL-7; and more generally, T cell receptor signaling.

The inventive compounds: (1) block IL-1β signal transduction through theType I receptor as shown, for example, by preventing IL-1β and IL-1βplus PDGF induction of proliferation of smooth muscle, endothelial andkidney mesangial cells; (2) suppress up-regulation of adhesion moleculesas shown, for example, by blocking VCAM in endothelial cells; (3)inhibit TNFα, LPS and IL-1β induced metalloproteases (an inflammationand cancer metasteses model); (4) block LPS, TNFα or IL-1β inducedsecondary cytokine production (for prevention and treatment of septicshock symptoms); (5) suppress T- and B-cell activation by antigen, forexample, IL-2 and IL-4; (6) inhibit mast cell activation byimmunoglobulin E (IgE); (7) are cytotoxic for transformed cells andtumor cell lines, yet not for normal cells at equivalent doses; and (8)block signaling by IL-2, IL-4, IL-6 and IL-7 on T- and B-cells.

The foregoing cellular results are the basis for the followingpharmacologic effects, including, but not limited to, protection andtreatment of endotoxic shock and sepsis induced by gram positive or gramnegative bacteria, inhibition and prevention of tumor cell growth andmetastatic spread, immunosuppression, treatment of autoimmune diseases,suppression of allograft reactions, stimulation of hair growth (e.g.,treatment of baldness or prevention of hair loss due to cytoreductivetherapies), and treatment of hyperproliferative skin disorders such aspsoriasis through reversal of an apoptotic process. The inventivecompounds are most useful to treat acute and chronic inflammatorydisease, suppress an immune response, treat cancer by having anapoptotic cytotoxic effect to transformed cells while having minimaltoxicity to rapidly proliferating cells at doses cytotoxic to tumorcells, prevent metastatic tumor growth through antiangiogenic andanti-adhesion properties, and treat or prevent an autoimmune disease andstimulate hair growth (when applied topically).

The inventive compounds also are useful as an adjuvant to inhibit toxicside effects of drugs whose side effects are mediated through thepresent second messenger pathway. Metalloproteases mediate tissue damagesuch as glomerular diseases of the kidney, joint destruction inarthritis, and lung destruction in emphysema, and play a significantrole in tumor metastases. Three examples of metalloproteases include a92 kD type V gelatinase induced by TNFα, IL-1β and PDGF plus b-FGF, a 72kD type IV collagenase that is usually constitutively produced andstimulated by TNFα or IL-1β and a stromelysin/PUMP-1 induced by TNFα andIL-1β. The inventive compounds can inhibit TNFα or IL-1β induction ofthe 92 kD type V gelatinase inducable metalloprotease. Moreover, theinventive compounds can reduce PUMP-1 activity induced by 100 U/ml ofIL-1β. Accordingly, the inventive compounds prevent induction of certainmetalloproteases induced by IL-1β or TNFα and are not involved withconstitutively produced proteases (e.g., 72 kD type IV collagenase)involved in normal tissue remodeling.

The inventive compounds inhibit IL-1 signal transduction, and aretherefore considered as IL-1 antagonists. A review article entitled"Mechanisms of Disease: The Role of Interleukin-1 in Disease" (Dinarelloet al., N. Engl. J. Med., Vol. 328, No. 2, pages 106-113, 1993)described the role of IL-1 as "an important rapid and direct determinantof disease." "In septic shock, for example, IL-1 acts directly on theblood vessels to induce vasodilatation through the rapid production ofplatelet activating factor and nitric oxide, whereas in autoimmunedisease it acts by stimulating other cells to produce cytokines orenzymes that then act on the target tissue." The article describes agroup of diseases that are mediated by IL-1, including sepsis syndrome,rheumatoid arthritis, inflammatory bowel disease, acute and myelogenousleukemia, insulin-dependent diabetes mellitus, atherosclerosis and otherdiseases including transplant rejection, graft versus host disease(GVHD), psoriasis, asthma, osteoporosis, periodontal disease, autoimmunethyroiditis, alcoholic hepatitis, premature labor secondary to uterineinfection and even sleep disorders. Since the inventive compounds areIL-1 antagonists, the inventive compounds are useful for treating all ofthe above-mentioned diseases.

For example, for sepsis syndrome, the mechanism of IL-1-induced shockappears to be the ability of IL-1 to increase the plasma concentrationsof small mediator molecules such as platelet activating factor,prostaglandin and nitric oxide. These substances are potent vasodilatorsand induce shock in laboratory animals. Blocking the action of IL-1prevents the synthesis and release of these mediators. In animals, asingle intravenous injection of IL-1 decreases mean arterial pressure,lowers systemic vascular resistance, and induces leukopenia andthrombocytopenia. In humans, the intravenous administration of IL-1 alsorapidly decreases blood pressure, and doses of 300 ng or more perkilogram of body weight may cause severe hypotension. The therapeuticadvantage of blocking the action of IL-1 resides in preventing itsdeleterious biologic effects without interfering with the production ofmolecules that have a role in homeostasis. The present inventivecompounds address the need identified by Dinarello et al. inhibitingIL-1 cellular signaling.

With regard to rheumatoid arthritis, Dinarello et al. state:"Interleukin-1 is present in synovial lining and synovial fluid ofpatients with rheumatoid arthritis, and explants of synovial tissue fromsuch patients produce IL-1 in vitro. Intraarticular injections ofinterleukin-1 induce leukocyte infiltration, cartilage breakdown, andperiarticular bone remodeling in animals. In isolated cartilage and bonecells in vitro, interleukin-1 triggers the expression of genes forcollagenases as well as phospholipases and cyclooxygenase, and blockingits action reduces bacterial-cell-wall-induced arthritis in rats."Therefore, the inventive compounds, as IL-1 antagonists, are useful totreat and prevent rheumatoid arthritis.

Inflammatory bowel disease, ulcerative colitis and Crohn's disease arecharacterized by infiltrative lesions of the bowel that containactivated neutrophils and macrophages. IL-1β can stimulate production ofinflammatory eicosanoids such as prostaglandin E₂ (PGE₂) and leukotrieneB₄ (LTB₄) and IL-8, an inflammatory cytokine withneutrophil-chemoattractant and neutrophil-stimulating properties. Tissueconcentrations of PGE₂ and LTB₄ correlate with the severity of diseasein patients with ulcerative colitis, and tissue concentrations of IL-1βand IL-8 are high in patients with inflammatory bowel disease.Therefore, an IL-1 antagonist, such as the inventive compounds, iseffective to treat inflammatory bowel disease.

With regard to acute and chronic myelogenous leukemia, there isincreasing evidence that IL-1 acts as a growth factor for such tumorcells. Therefore, the inventive compounds are effective to preventdisease deterioration for acute and chronic myelogenous leukemias.

IDDM is considered to be an autoimmune disease with destruction of betacells in the islets of Langerhans mediated by immunocompetent cells.Islets of animals with spontaneously occurring IDDM (e.g., BB rats orNOD mice) have inflammatory cells that contain IL-1β. Therefore, theinventive compounds are useful for the prevention of and treatment ofIDDM.

IL-1β also plays a role in the development of atherosclerosis.Endothelial cells are a target of IL-1β. IL-1β stimulates proliferationof vascular smooth muscle cells. Foam cells isolated from fatty arterialplaques from hypercholesterolemic rabbits contain IL-1β and IL-1βmessenger RNA. The uptake of peripheral blood monocytes results ininitiation of IL-1β production by these cells. IL-1β also stimulatesproduction of PDGF. Taken together, IL-1β plays a part in thedevelopment of atherosclerotic lesions. Therefore, an IL-1β antagonist,such as the inventive compounds is useful in preventing and treatingatherosclerosis.

DAG and PA are up-regulated in oncogenically transformed cells. Forexample, activating ras mutations result in increased generation of DAGon stimulation with mitogens. In non-transformed renal mesangial cells,IL-1β stimulation increased PLA2 and LPAAT activation, resulting ingeneration of sn-2 unsaturated PA and subsequent hydrolysis to DAG byphosphatidate phosphohydrolase. The ras transformation in NIH/3T3 cellsup-regulates serum-stimulated generation of DAG and PA. The specificspecies of DAG stimulated by serum is dioleoyl and for PA, dilinoleoyland dioleoyl. This upregulation occurs over 4-12 hours and pretreatmentof cells with an compound blocks generation of these phospholipid secondmessengers. The inhibition occurs either through suppressing thegeneration of PA de novo from lysoPA, or through inhibition of one orboth arms of the Lands cycle. Therefore ras transformation mediates anup-regulation of specific PA species through indirect stimulation ofPLA2 and/or LPAAT activity. The inventive compounds inhibit theconversion of the upregulated lysoPA to PA and subsequently blockcellular phenotypic changes induced by PA/DAG in the membrane.

The ability of the inventive compounds to inhibit generation ofunsaturated phospholipids is mirrored by the ability of compounds toinhibit proliferation and tumorogenicity of ras-transformed cells invitro and in vivo. This inhibition is reversible and is not associatedwith significant cytotoxicity.

Excessive or unregulated TNFα production is implicated in mediating orexacerbating a number of diseases including rheumatoid arthritis,rheumatoid spondylitis, osteoarthritis, gouty arthritis and otherarthritic conditions, sepsis, septic shock, endotoxic shock, gramnegative sepsis, toxic shock syndrome, adult respiratory distresssyndrome, cerebral malaria, chronic pulmonary inflammatory disease,silicosis, pulmonary sarcoidosis, bone resorption diseases, reperfusioninjury, graft versus host reaction, allograft rejections, fever,myalgias due to infection such as influenza, cachexia secondary toinfection, AIDS or malignancy, other viral infections (e.g., CMV,influenza, adenovirus, herpes family), keloid formation, scar tissueformation, Crohn's disease, ulcerative colitis, or pyresis. Thecompounds or pharmaceutically acceptable salts thereof can be used inthe manufacture of a medicament for the prophylactic or therapeutictreatment of any disease state in a human or other mammal, which isexacerbated or signaled through the present second messenger cellularphospholipid-based signaling pathway and by excessive or unregulatedproduction of "first messenger" inflammatory cytokines such as TNFα orIL-1β. With regard to TNFα first messenger signaling, there are severaldisease states in which excessive or unregulated TNFα production bymonocytes/macrophages is implicated in exacerbating or causing thedisease. These include, for example, neurodegenerative diseases such asAlzheimers disease, endotoxemia or toxic shock syndrome (Tracey et al.,"Anti-cachectin/TNF Monoclonal Antibodies Prevent Septic Shock DuringLethal Bacteraemia," Nature, Vol. 330, pages 662-664, Dec. 17, 1987 andHinshaw et al., "Survival of Primates in LD₁₀₀ Septic Shock FollowingTherapy With Antibody to Tumor Necrosis Factor (TNFα)," Circ. Shock,Vol. 30, pages 279-292, 1990); cachexia (Dezube et al., "Pentoxifyllineand Wellbeing in Patients with Cancer," The Lancet, page 662, Mar. 17,1990), and adult respiratory distress syndrome (Miller et al., "TumourNecrosis Factor in Bronchopulmonary Secretions of Patients with AdultRespiratory Distress Syndrome," The Lancet, pages 712-713, Sep. 23,1989). The compounds may be used topically in the treatment ofprophylaxis of topical disease states mediated or exacerbated byexcessive TNFα or IL-1β, such as viral infections (herpes or viralconjunctivitis), psoriasis, fungal or yeast infections (ringworm,athletes foot, vaginitis, dandruff, etc.) or other dermatologichyperproliferative disorders. High TNFα levels have been implicated inacute malaria attacks (Grau et al., "Tumor Necrosis Factor and DiseaseSeverity in Children with Falciparum Malaria," N. Engl. J. Med., Vol.320, No. 24, pages 1586-1591, Jun. 15, 1989), chronic pulmonaryinflammatory diseases such as silicosis and asbestosis (Piguet et al.,"Requirement of Tumour Necrosis Factor for Development of Silica-inducedPulmonary Fibrosis," Nature, Vol. 344, pages 245-247, Mar. 15, 1990, andBissonnette et al., "Pulmonary Inflammation and Fibrosis in a MurineModel of Asbestosis and Silicosis," Inflammation, Vol. 13, No. 3, pages329-339, 1989), and reperfusion injury (Vedder et al., "Inhibition ofLeukocyte Adherence by Anti-CD18 Monoclonal Antibody AttenuatesReperfusion Injury in the Rabbit Ear, Proc. Natl. Acad. Sci. USA, Vol.87, pages 2643-2646, April 1990).

The invention includes methods for treating or preventing clinicalsymptoms of various disease states or reducing toxicity of othertreatments by inhibiting cellular signaling through the second messengerpathway. Disease state or treatment-induced toxicity are selected fromthe group consisting of proliferation of tumor cells in response to anactivated oncogene; hematocytopenia caused by cytoreductive therapies;autoimmune diseases caused by a T-cell response or a B-cell response andantibody production; septic shock; resistance of mesenchymal cells toTNFα; proliferation of smooth muscle cells endothelial cells,fibroblasts and other cell types in response to growth factors, such asPDGF, FGF, EGF and VEGF (i.e., atherosclerosis, restenosis, stroke, andcoronary artery disease); human immunodeficiency virus infection (AIDSand AIDS related complex); proliferation of kidney mesangial cells inresponse to IL-1β, MIP-1α, PDGF or FGF; inflammation; kidney glomerularor tubular toxicity in response to cyclosporin A or amphotericin Btreatment; organ toxicity (e.g., gastrointestinal or pulmonaryepithelial) in response to a cytoreductive therapy (e.g., cytotoxic drugor radiation); enhancing antitumor effects of non-alkylating antitumoragents; allergies in response to inflammatory stimuli (e.g., TNFα, IL-1βand the like) characterized by production of cell surfacemetalloproteases or by degranulation of mast cells and basophils inresponse to IgE, bone diseases caused by overproduction ofosteoclast-activating factor (OAF) by osteoclasts, CNS diseases causedby reduced signal transduction of the neurotransmitters epinephrine andacetylcholine, and combinations thereof. The compounds are also usefulas antimicrobial agents to directly treat fungal or yeast infections andto indirectly treat bacterial or viral infections through an immunestimulation and pro-hematopoietic effect.

In summary, the compounds and pharmaceutical compositions thereofexhibit some of the following activities: (1) are cytotoxic to tumorcells at doses that are not cytotoxic to normal cells; (2) suppressactivation of T-cells by antigen or IL-2 stimulation; (3) suppressactivation of monocyte/macrophage cells by endotoxin, TNFα, IL-1β orGM-CSF stimulation; (4) suppress antibody production of B-cells inresponse to an antigen, IL-4 or CD40 ligand; (5) inhibit theproliferation of smooth muscle cells in response to growth factorscapable of stimulating said proliferation (e.g., by inhibitingangiogenesis or preventing atherosclerosis and restenosis or repertusioninjury); (6) lower expression of adhesion molecules induced by enhancersthereof; (7) inhibit the proliferation of kidney mesangial cells inresponse to stimulation by IL-1β and/or MIP-1α and/or PDGF and/or FGF;(8) enhance the resistance of kidney glomerular or tubular cells tocyclosporin A or amphotericin B; (9) prevent the release of MIP-1α byIL-1α, TNFα, or endotoxin stimulated monocytes and macrophages; (10)prevent the release of platelet activating factor by IL-1β, TNFα, orendotoxin treated megakaryocytes, fibroblastic cells, and macrophages;(11) prevent the down-regulation of receptors for cytokines in TNFα-treated hematopoietic progenitor cells; (12) suppress the production ofmetalloproteases in IL-1β-stimulated or TNFα-stimulated glomerularepithelial cells or synovial cells; (13) enhance the resistance ofgastrointestinal or pulmonary epithelial cells to cytotoxic drugs orradiation; (14) enhance the antitumor effect of a non-alkylatingantitumor agent; (15) to inhibit the production of osteoclast activatingfactor in response to IL-1β; (16) inhibit degranulation in response toIgE; (17) enhance the release of adrenergic neural transmitters,dopamine, norepinephrine, or epinephrine, or the neurotransmitter,acetylcholine; (18) modulate the post-synaptic "slow current" effects ofthe adrenergic neurotransmitters dopamine, epinephrine, ornorepinephrine, or the neurotransmitter acetylcholine; (19) suppresssignaling by neurotransmitters including acetyl choline, leuenkephalinand seretonin; or (20) increase seizure threshold.

The compounds are also useful to raise the seizure threshold, tostabilize synapses against neurotoxins such as strychnine, to potentiatethe effect of anti-Parkinson drugs such as L-dopa, to potentiate theeffects of soporific compounds, to relieve motion disorders resultingfrom administration of tranquilizers, and to diminish or prevent neuronoverfiring associated with progressive neural death following cerebralvascular events such as stroke. In addition, the compounds of theinvention are useful in the treatment of norepinephrine-deficientdepression and depressions associated with the release of endogenousglucocorticoids, to prevent the toxicity to the central nervous systemof dexamethasone or methylprednisolone, and to treat chronic painwithout addiction to the drug. Further, the compounds of the inventionare useful in the treatment of children with learning and attentiondeficits and generally improve memory in subjects with organic deficits,including Alzheimer's patients.

Compounds of the Invention

The invention provides for a class of compounds that are effectivetherapeutic agents to inhibit specific inflammatory and proliferativecellular signaling events. The compounds include resolved enantiomersand/or diastereomers, hydrates, salts, solvates and mixtures thereof,the compound having a straight or branched aliphatic hydrocarbonstructure of formula I: ##STR5##

In formula I, n is an integer from one to four and m is an integer fromfour to twenty. Independently, R₁ and R₂ are hydrogen, a straight orbranched chain alkyl, alkenyl or alkynyl of up to twenty carbon atoms inlength or --(CH₂)_(w) R₅. If R₁ or R₂ is --(CH₂)_(w) R₅, w may be aninteger from one to twenty and R₅ may be an hydroxyl, halo, C₁₋₈ alkoxylgroup or a substituted or unsubstituted carbocycle or heterocycle.Alternatively, R₁ and R₂ may jointly form a substituted orunsubstituted, saturated or unsaturated heterocycle having from four toeight carbon atoms, N being a hetero atom of the resulting heterocycle.R₃ may be either hydrogen or C₁₋₃. Preferred compounds may have one ofR₁ or R₂ and R₃ that form a substituted or unsubstituted linking carbonchain, having from one to four carbon atoms. This R₁ /R₃ or R₂ /R₃linking chain will join the O and N in a cyclic structure, an integersum equal to n+a number of carbon atoms in the linking carbon chainbeing less than six.

In the compounds, a total sum of carbon atoms comprising R₁ or R₂,(CH₂)_(n) and (CH₂)_(m) does not exceed forty. R₄ is a terminal moietycomprising a substituted or unsubstituted, oxidized or reduced ringsystem, the ring system having a single ring or two to three fusedrings, a ring comprising from three to seven ring atoms. However, if R₄is phthalimide, m of formula I is not less than five.

In preferred compounds of the invention which have a general structureof formula I, R₅ may be hydroxy, chloro, fluoro, bromo, or C₁₋₆ alkoxy,or a substituted or unsubstituted, saturated or unsaturated heterocyclehaving from four to seven carbon atoms, more preferably, a mono-, di- ortri-substituted carbocycle or heterocycle. In the compounds, (CH₂)_(m)may be unsubstituted, or more preferably, (CH₂)_(m) is substituted by ahalogen atom, an hydroxyl group, or substituted or unsubstitutedC.sub.(1-10) alkoxyl, C.sub.(1-10) alkyl, C.sub.(2-10) alkenyl orC.sub.(1-10) alkynyl group. Substituents of the R₁ /R₃ or R₂ /R₃ linkingchain may include, without limitation, a C(₁₋₄) alkyl, C(₂₋₄) alkenyl,hydroxyl, carbonyl, amino, thio, thiol, thiocarbonyl and imino group ora single atom, such as, for example, chlorine, bromine, fluorine andoxygen

In the compounds comprising a non-cyclic, terminal moiety, the terminalmoiety may include, without limitation, an acetamidyl, amidyl, aminyl,amino acid (one or two), carbonyl, carboxyl, alkoxylcarbonyl, halo,hydro, hydroxyl, glutaric acid, alkoxyl, phosphatyl, phosphonatyl,sulfatyl, sulfonatyl, sulfonyl, sulfoxidyl, thio or thiolalkoxylcarbonylgroup or a simple ionic functional group. A terminal moiety amino acidmay be one or more of the following: alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine and valine, and are preferably a morehydrophobic amino acid such as leucine or isoleucine. In preferredcompounds of the invention, the non-cyclic terminal moiety may be adipeptide comprising two amino acids selected from the foregoingexemplary list. A preferred halo group may include, but is not limitedto bromo, chloro, fluoro or iodo.

In more preferred compounds, the terminal moiety ring system may besaturated, but alternatively, preferred compounds have a ring systemterminal moiety having at least one unsaturated carbon-carbon doublebond. In more preferred compounds of the invention, ring systemsubstituents may include, but are not intended to be limited to, C(₁₋₄)alkyl, C(₂₋₄) alkenyl, hydroxyl, carbonyl, amino, thio, thiol,thiocarbonyl and imino group or a single atom. Single atomscorresponding to ring substituents may include, but are not intended tobe limited to, chlorine, bromine, fluorine and oxygen.

The compounds may have ring systems, in which all ring atoms are carbonatoms. Preferred compounds, in which all ring atoms are carbon atoms,may have ring systems that include, but are not intended to be limitedto, one of the following groups: phenyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl,cycloheptenyl, biscyclooctyl, indanyl, indenyl, decalinyl, resorcinolyl,tetralinyl, α-tetralonyl, 1-indanonyl, cyclohexanedionyl orcyclopentanedionyl.

In more preferred compounds of the invention, at least one ring atom ofthe ring system may be other than carbon. For preferred compounds thathave a ring system terminal moiety, having at least one ring atom whichis other than carbon, then the total number of non-carbon ring atomsshould not exceed a value "X," specified by the equation: X=total numberof ring atoms-1. Non-carbon ring atoms may include, for example, atomssuch as nitrogen, oxygen, sulfur and phosphorus.

Terminal moiety ring systems which have at least one ring atom that isother than carbon may include, for example, ring systems that have threeor four atoms in at least one ring of the system. Preferred ring systemshaving at least one non-carbon atom and at least one ring which hasthree or four atoms may include, but are not intended to be limited to:azetidinedionyl; azetidinonyl; azetidinyl; aziridinonyl; aziridinyl;azirinyl; diaziridinonyl; diaziridinyl; diazirinyl; dioxetanyl;dioxiranyl; dithietanyl; episulfonyl; lactamyl; lactonyl; oxathietanyl;oxathiiranyl; oxaziranyl; oxaziridinyl; oxetananonyl; oxetanonyl;oxetanyl; oxiranyl; sultamyl; sultinyl; sultonyl; thiazetidinyl;thiaziridinyl; thietanyl or thiiranyl.

Alternatively, preferred compounds may have terminal moieties that haveat least one ring having at least five ring atoms, at least one of theat least five ring atoms being other than carbon. In these preferredcompounds, the at least five-atom ring system may include, but is notintended to be limited to, one of the following substituted orunsubstituted groups: adeninyl; alloxanyl; alloxazinyl; anthracenyl;anthrenyl; azapinyl; azapurinyl; azinyl; azolyl; barbituric acid;biotinyl; chromylenyl; cinnolinyl; coumarinyl; coumaronyl; depsidinyl;diazepinyl; diazinyl; diazocinyl; dioxadiazinyl; dioxanyl; dioxenyl;dioxepinyl; dioxinonyl; dioxolanyl; dioxolonyl; dioxolyl;dioxanthylenyl; enantholactamyl; flavanyl; flavinyl; flavonyl;fluoranyl; fluorescienyl; furandionyl; faranochromanyl; furanonyl;furanyl; furazanyl; furoxanyl; guaninyl; hydroquinolinyl;imidazolethionyl; imidazolinyl; imidazolonyl; imidazolyl; indolizidinyl;indolizinyl; indolonyl; indolyl; isatinyl; isatogenyl; isoindolinyl;isoquinolinyl; isothiazolyl; isoxazolyl; lactamyl; lactonyl; lumazinyl;naphthacenyl; naphthalenyl; oroticyl; oxadiazinyl; oxadiazolyl;oxathianyl; oxathiazinonyl; oxathiolanyl; oxatriazolyl; oxazinonyl;oxazolidinonyl; oxazolidinyl; oxazolinonyl; oxazolinyl; oxazolonyl;oxazolyl; oxolenyl; pentazinyl; pentazolyl; petrazinyl; phthalimidyl;phthalonyl; piperazindionyl; piperazinodionyl; piperazinyl; piperidinyl;piperidonyl; prolinyl; prylenyl; pteridinyl; pterinyl; purinyl;pyradinyl; pyranoazinyl; pyranoazolyl; pyranonyl; pyranyl; pyrazinyl;pyrazolidinyl; pyrazolidonyl; pyrazolinonyl; pyrazolinyl; pyrazolonyl;pyrazolyl; pyrenyl; pyridazinyl; pyridazonyl; pyridinyl; pyrimidinyl;pyrimidionyl; pyronyl; pyrrolidinyl; pyrrolyl; quinazolidinyl;quinazolinonyl; quinazolinyl; quinolinyl; quinolizinyl; quinonyl;quinoxalinyl; quinuclidinyl; rhodaminyl; spirocoumaranyl; succinimidyl;sulfolanyl; sulfolenyl; sultamyl; sultinyl; sultonyl; sydononyl;tetraoxanyl; tetrazepinyl; tetrazinyl; tetrazolyl; tetronyl;thiazepinyl; thiazinyl; thiazolyl; thiepinyl; thiolanyl; thiolenyl;thiolyl; thiophenyl; thyminyl; triazepinonyl; triazepinyl; triazinyl;triazolinyl; triazolyl; trioxanyl; trithianyl; trixolanyl; trizinyl;tropanyl; uracilyl; xanthenyl; xanthinyl; xanthonyl; xanthydrolyl orxylitolyl.

For compounds having a preferred ring system terminal moiety having atleast one ring with one hetero atom, a ring system may include one ofthe following: acridinyl; acridonyl; alkylpyridinyl; anthraquinonyl;ascorbyl; azaazulenyl; azabenzanthracenyl; azabenzanthrenyl;azabenzophenanthrenyl; azachrysenyl; azacyclazinyl; azaindolyl;azanaphthacenyl; azanaphthalenyl; azapyrenyl; azatriphenylenyl;azepinyl; azinoindolyl; azinopyrrolyl; benzacridinyl; benzazapinyl;benzofuryl; benzonaphthyridinyl; benzopyranonyl; benzopyranyl;benzopyronyl; benzoquinolinyl; benzoquinolizinyl; benzothiepinyl;benzothiophenyl; benzylisoquinolinyl; biotinyl; bipyridinyl;butenolidyl; butyrolactonyl; caprolactamyl; carbazolyl; carbolinyl;catechinyl; chromenopyronyl; chromonopyranyl; coumarinyl; coumaronyl;decahydroquinolinyl; decahydroquinolonyl; diazaanthracenyl;diazaphenanthrenyl; dibenzazepinyl; dibenzofuranyl; dibenzothiophenyl;dichromylenyl; dihydrofuranyl; dihydroisocoumarinyl;dihydroisoquinolinyl; dihydropyranyl; dihydropyridinyl;dihydropyridonyl; dihydropyronyl; dihydrothiopyranyl; diprylenyl;dioxanthylenyl; enantholactamyl; flavanyl; flavonyl; fluoranyl;fluorescienyl; furandionyl; furanochromanyl; furanonyl;furanoquinolinyl; furanyl; furopyranyl; furopyronyl; heteroazulenyl;hexahydropyrazinoisoquinolinyl; hydrofuranyl; hydrofurnanonyl;hydroindolyl; hydropyranyl; hydropyridinyl; hydropyrrolyl;hydroquinolinyl; hydrothiochromenyl; hydrothiophenyl; indolizidinyl;indolizinyl; indolonyl; isatinyl; isatogenyl; isobenzofurandionyl;isobenzofuranyl; isochromanyl; isoflavonyl; isoindolinyl;isoindolobenzazepinyl; isoindolyl; isoquinolinyl; isoquinuclidinyl;lactamyl; lactonyl; maleimidyl; monoazabenzonaphthenyl; naphthalenyl;naphthimidazopyridinedionyl; naphthindolizinedionyl;naphthodihydropyranyl; naphthofuranyl; naphthothiophenyl;naphthyridinyl; oxepinyl; oxindolyl; oxolenyl; perhydroazolopyridinyl;perhydroindolyl; phenanthraquinonyl; phenanthridinyl; phenanthrolinyl;phthalideisoquinolinyl; phthalimnidyl; phthalonyl; piperidinyl;piperidonyl; prolinyl; pyradinyl; pyranoazinyl; pyranoazolyl;pyranopyrandionyl; pyranopyridinyl; pyranoquinolinyl; pyranopyradinyl;pyranyl; pyrazolopyridinyl; pyridinethionyl; pyridinonaphthalenyl;pyridinopyridinyl; pyridinyl; pyridocolinyl; pyridoindolyl;pyridopyridinyl; pyridopyrimidinyl; pyridopyrrolyl; pyridoquinolinyl;pyronyl; pyrrocolinyl; pyrrolidinyl; pyrrolizidinyl; pyrrolizinyl;pyrrolodiazinyl; pyrrolonyl; pyrrolopyrimidinyl; pyrroloquinolonyl;pyrrolyl; quinacridonyl; quinolinyl; quinolizidinyl; quinolizinyl;quinolonyl; quinuclidinyl; rhodaminyl; spirocoumaranyl; succinimidyl;sulfolanyl; sulfolenyl; tetrahydrofuranyl; tetrahydroisoquinolinyl;tetrahydropyranyl; tetrahydropyridinyl; tetrahydrothiapyranyl;tetrahydrothiophenyl; tetrahydrothiopyranonyl; tetrahydrothiopyranyl;tetronyl; thiabenzenyl; thiachromanyl; thiadecalinyl; thianaphthenyl;thiapyranyl; thiapyronyl; thiazolopyridinyl; thienopryidinyl;thienopyrrolyl; thienothiophenyl; thiepinyl; thiochromenyl;thiocoumarinyl; thiophenyl; thiopyranyl; triazaanthracenyl;triazinoindolyl; triazolopyridinyl; tropanyl; xanthenyl; xanthonyl orxanthydrolyl.

In addition, compounds that have a preferred ring system terminal moietyhaving at least one ring with two hetero atoms, the ring system mayinclude: adeninyl; alloxanyl; alloxazinyl; anthranilyl;azabenzanthrenyl; azabenzonaphthenyl; azanaphthacenyl; azaphenoxazinyl;azapurinyl; azinyl; azoloazinyl; azolyl; barbituric acid; benzazinyl;benzimidazolethionyl; benzimidazolonyl; benzimidazolyl;benzisothiazolyl; benzisoxazolyl; benzocinnolinyl; benzodiazocinyl;benzodioxanyl; benzodioxolanyl; benzodioxolyl; benzopyridazinyl;benzothiazepinyl; benzothiazinyl; benzothiazolyl; benzoxazinyl;benzoxazolinonyl; benzoxazolyl; cinnolinyl; depsidinyl;diazaphenanthrenyl; diazepinyl; diazinyl; dibenzoxazepinyl;dihydrobenzimidazolyl; dihydrobenzothiazinyl; dihydrooxazolyl;dihydropyridazinyl; dihydropyrimidinyl; dihydrothiazinyl; dioxanyl;dioxenyl; dioxepinyl; dioxinonyl; dioxolanyl; dioxolonyl;dioxopiperazinyl; dipyrimidopyrazinyl; dithiolanyl; dithiolenyl;dithiolyl; flavinyl; furopyrimidinyl; glycocyamidinyl; guaninyl;hexahydropyrazinoisoquinolinyl; hexahydropyridazinyl; hydantoinyl;hydroimidazolyl; hydropyrazinyl; hydropyrazolyl; hydropyridazinyl;hydropyrimidinyl; imidazolinyl; imidazolyl; imidazoquinazolinyl;imidazothiazolyl; indazolebenzopyrazolyl; indoxazenyl; inosinyl;isoalloxazinyl; isothiazolyl; isoxazolidinyl; isoxazolinonyl;isoxazolinyl; isoxazolonyl; isoxazolyl; lumazinyl; methylthyminyl;methyluracilyl; morpholinyl; naphthimidazolyl; oroticyl; oxathianyl;oxathiolanyl; oxazinonyl; oxazolidinonyl; oxazolidinyl; oxazolidonyl;oxazolinonyl; oxazolinyl; oxazolonyl; oxazolopyrimidinyl oxazolyl;perhydrocinnolinyl; perhydropyrroloazinyl; perhydropyrrolooxazinyl;perhydropyrrolothiazinyl; perhydrothiazinonyl; perimidinyl; phenazinyl;phenothiazinyl; phenoxathiinyl; phenoxazinyl; phenoxazonyl;phthalazinyl; piperazindionyl; piperazinodionyl; polyquinoxalinyl;pteridinyl; pterinyl; purinyl; pyrazinyl; pyrazolidinyl; pyrazolidonyl;pyrazolinonyl; pyrazolinyl; pyrazolobenzodiazepinyl; pyrazolonyl;pyrazolopyridinyl; pyrazolopyrimidinyl; pyrazolotriazinyl; pyrazolyl;pyridazinyl; pyridazonyl; pyridopyrazinyl; pyridopyrimidinyl;pyrimidinethionyl; pyrimidinyl; pyrimidionyl; pyrimidoazepinyl;pyrimidopteridinyl; pyrrolobenzodiazepinyl; pyrrolodiazinyl;pyrrolopyrimidinyl; quinazolidinyl; quinazolinonyl; quinazolinyl;quinoxalinyl; sultamyl; sultinyl; sultonyl; tetrahydrooxazolyl;tetrahydropyrazinyl; tetrahydropyridazinyl; tetrahydroquinoxalinyl;tetrahydrothiazolyl; thiazepinyl; thiazinyl; thiazolidinonyl;thiazolidinyl; thiazolinonyl; thiazolinyl; thiazolobenzimidazolyl;thiazolyl; thienopyrimidinyl; thiazolidinonyl; thyminyl;triazolopyrimidinyl; uracilyl; xanthinyl; or xylitolyl.

Terminal ring systems having at least one ring having three hetero atomsmay include, but are not intended to be limited to, one of the followingring systems: azabenzonaphthenyl; benzofuroxanyl; benzothiadiazinyl;benzotriazepinonyl; benzotriazolyl; benzoxadizinyl; dioxadiazinyl;dithiadazolyl; dithiazolyl; furazanyl; furoxanyl; hydrotriazolyl;hydroxytrizinyl; oxadiazinyl; oxadiazolyl; oxathiazinonyl; oxatriazolyl;pentazinyl; pentazolyl; petrazinyl; polyoxadiazolyl; sydononyl;tetraoxanyl; tetrazepinyl; tetrazinyl; tetrazolyl; thiadiazinyl;thiadiazolinyl; thiadiazolyl; thiadioxazinyl; thiatriazinyl;thiatriazolyl; thiatriazolyl; triazepinyl; triazinoindolyl; triazinyl;triazolinedionyl; triazolinyl; triazolyl; trioxanyl; triphenodioxazinyl;triphenodithiazinyl; trithiadiazepinyl; trithianyl; or trixolanyl.

In these compounds, the most preferred ring systems include, forexample, dimethylxanthinyl, methylxanthinyl, phthalimidyl,homophthalimidyl, methylbenzoyleneureayl, quinazolinonyl,octylcarboxamidobenzenyl, methylbenzamidyl,methyldioxotetrahydropteridinyl, glutarimidyl, piperidonyl,succinimidyl, dimethoxybenzenyl, methyldihydrouracilyl, methyluracilyl,methylthyminyl, piperidinyl, dihydroxybenzenyl, or methylpurinyl, evenmore preferably, methylxanthinyl, dimethylxanthinyl or a derivativethereof. The most preferred compounds may also have a ring-systemterminal moiety that has at least one substituent bonded to at least onering of the ring system, the at least one substituent being bonded to acarbon ring atom of the at least one ring by an sp bond, in which thecarbon ring atom is adjacent to a hetero atom of the ring. Also, in themost preferred embodiments of the compounds, ring-system terminalmoieties, having at least one hetero atom, may be linked to --(CH₂)_(m)of formula I by a bond between the at least one hetero atom and--(CH₂)_(m).

The compounds may include resolved enantiomers and/or diastereomers,hydrates, salts, solvates and mixtures thereof of compounds that have astraight or branched aliphatic hydrocarbon structure of formula II:##STR6##

In the above formula II, n, m, R₃, and R₄ are defined as provided informula I above. R₆ and R₇ are hydrogen, a straight or branched chainalkane, alkene or alkyne of up to twenty carbon atoms in length, or--(CH₂)_(x) R₈, at least one of R₆ or R₇ being --(CH₂)_(x) R₈. Informula II, x is an integer from zero to fourteen and R₈ is a moietyhaving a general structure as provided in formula III ##STR7##

In formula III above, m, R₃, and R₄ are defined as provided in formula Iabove. Z is N or CH and p is an integer from zero to four. R₉ is H or astraight or branched chain alkane, alkene or alkyne of up to twentycarbon atoms in length.

The invention provides a pharmaceutical composition comprising ancompound and a pharmaceutically acceptable excipient. The pharmaceuticalcomposition may be formulated for oral, parenteral or topicaladministration to a patient.

The invention further provides a pharmaceutical composition comprisingan compound and a pharmaceutically acceptable excipient, thepharmaceutical composition being formulated for oral, parenteral ortopical administration to a patient. A pharmaceutical composition mayalternatively comprise one or a plurality of compounds and apharmaceutically acceptable carrier or excipient. Treatment ofindividuals with an compound or pharmaceutical composition may includecontacting with the compound in vitro culture, in an extracorporealtreatment, or by administering (oral, parenteral or topical) thecompound or pharmaceutical composition to a subject whose cells are tobe treated.

Synthesis of the Inventive Compounds

The invention includes a method for preparing the inventive compounds.An exemplary method for preparing the inventive compounds is discussedbelow and in the following examples. In a synthesis according to theinvention, a compound containing a desired terminal group (intended as a"terminal moiety" in compounds of the invention) undergoes a reaction toproduce an anion of the terminal moiety-containing compound.Subsequently, the resulting anion is reacted with a substituted olefinto displace a targeted functional group on the olefin, resulting in anintermediate product. A predetermined amount of a terminalmoiety-containing compound is reacted with a suitable base, a solventand a substituted olefin, the substituted olefin having at least oneother functional group which may be substituted in a displacementreaction by the desired terminal moiety-containing compound.

Preferred bases include, but are not limited to, sodium hydride, sodiumamide, sodium alkoxide, lithium hydride, potassium hydride, lithiumamide, sodium amide and potassium amide. An especially preferred base issodium hydride. Preferred solvents may be dimethylsulfoxide,dimethylformamide, or an alcohol. Exemplary preferred alcohols include,but are not limited to, methanol, ethanol or isopropanol. Anysubstituted olefin comprising a chain structure of the inventivecompounds may be used in the reaction according to the invention.Preferred olefins may be ω-substituted olefins. Preferred substitutedolefins include, but are not limited to halo-substituted olefins.

The intermediate product, having a composite structure of the terminalmoiety-containing compound and substituted olefin, may subsequently beconverted to a corresponding epoxide. In the method according to theinvention, the intermediate product may be reacted with an organicperacid to obtain a desired epoxide. Preferred, exemplary organicperacids include 3-chloroperoxybenzoic acid, peracetic acid andtrifluoroperacetic acid. An especially preferred peracid is3-chloroperoxybenzoic acid.

Alternatively, the intermediate product may be converted first to acorresponding diol by reacting the intermediate product with a suitableoxidizing agent. Preferred oxidixing agents include, but are not limitedto, osmium tetroxide. Preferred oxidizing agents, such as osmiumtetroxide may require a catalytic amount of the oxidizing agent in thepresence of a regenerating agent. Exemplary, regenerating agents may be4-methylmorpholine-N-oxide and trimethylamine-N-oxide. An especiallypreferred regenerating agent is 4-methylmorpholine-N-oxide. In asubsequent halogenation reaction, the resulting diol is converted to ahaloester using a halogenating agent in the presence of an organic acid.Exemplary halogenating agents include hydrogen bromide and hydrogenchloride. Preferred organic acids may be acetic acid and propionic acid.The resulting haloester is subsequently reacted with a basicester-hydrolyzing reagent to obtain a desired epoxide product. Preferredester-hydrolyzing agents include, but are not limited to metal alkoxidesand metal hydroxides. Especially preferred metal alkoxides are sodiummethoxide, ethoxide, isopropoxide and pentoxide. A preferred metalhydroxide is sodium hydroxide.

A final step in the inventive method is preparation of the desiredinventive compound from a terminal moiety-containing epoxide,synthesized in the foregoing procedure. The final step may beaccomplished by either of two preferred methods. In a first method, theterminal moiety-containing epoxide is heated in the presence of asubstituted or unsubstituted amine having functional groups which arepresent in the final inventive compound. Preferred amine functionalgroups are disclosed above.

A second method comprises reacting the unsubstituted or substitutedamine with the terminal moiety-containing epoxide and a reactionactivator in a solvent. Exemplary reaction activators include lithiumperchlorate. Preferred solvents include solvents for reactionspreviously discussed herein.

Preferred compounds of the invention include, but are not intended to belimited to, both R and S enantiomers and racemic mixtures of thefollowing compounds:

    __________________________________________________________________________    Compound No.                                                                         Compound Name                       Chemical Structure                 __________________________________________________________________________    1      N-(9-Octylamino-8-hyroxynonyl)phthalimide                               ##STR8##                                                                     2      N-(11-Octylamino-10-hydroxyundecyl)homophthalimide                      ##STR9##                                                                     3      1-(5-hydroxy-6-(N-benzyl)aminohexyl)-3-methylbenzoyleneurea             ##STR10##                                                                    4      3-(11,10-Oxidoundecyl)quinazoline-4(3H)-one                             ##STR11##                                                                    5      N.sup.2 -(5-hydroxy-6-(N.sup.3 -propyl)aminohexyl)-(N.sup.1                   -propyl)glutaric acid                                                   ##STR12##                                                                    6      2-(11-Octylamino-10-hydroxyundecylcarboxamido)-octylcarboxamidobenz           yl                                                                      ##STR13##                                                                    7      1-Octylamino-2,11-undecadiol                                            ##STR14##                                                                    8      1-(9-Octylamino-8-hydroxynonyl)-3-methylxanthine                        ##STR15##                                                                    9      1-(9-Tetradecylamino-8-hydroxynonyl)-3-methylxanthine                   ##STR16##                                                                    10     1-(11-Octylamino-10-hydroxyundecyl)-3-methylxanthine                    ##STR17##                                                                    11     7-(11-Octylamino-10-hydroxyundecyl)-1,3-dimethylxanthine                ##STR18##                                                                    12     1-(11,10-Octylamino-10-hydroxyundecyl)-1-methyl-2,4-dioxotetrahydro           pteridine                                                               ##STR19##                                                                    13     1-(5-hydroxy-6-(N-benzyl)aminohexyl)-3,7-dimethylxanthine               ##STR20##                                                                    14     1-(5-hydroxy-6-(N-propyl)aminohexyl)-3,7-dimethylxanthine               ##STR21##                                                                    15     N-(11-Ocytlamino-10-hydroxyundecyl)glutarimide                          ##STR22##                                                                    16     N-(11-Octylamino-10-hydroxyundecyl)-2-piperidone                        ##STR23##                                                                    17     N-(11-Octylamino-10-hydroxyundecyl)succinimide                          ##STR24##                                                                    18     2-(11-Octylamino-10-hydroxyundecyl)-1,3-dimethoxybenzene                ##STR25##                                                                    19     3-(5-hydroxy-6-(N-propyl)aminohexyl)-1-methyluracil                     ##STR26##                                                                    20     3-(9-Octylamino-8-hydroxynonyl)-1-methyluracil                          ##STR27##                                                                    21     3-(11-Octylamino-10-hydroxyundecyl)-1-methyluracil                      ##STR28##                                                                    22     3-(11-Octylamino-10-hydroxyundecyl)-1-methyldihydrouracil               ##STR29##                                                                    23     3-(9-Octylamino-8-hydroxynonyl)-1-methylthymine                         ##STR30##                                                                    24     3-(5-hydroxy-6-(N-undecyl)aminohexyl)-1-methylthymine                   ##STR31##                                                                    25     3-(11-Octylamino-10-hydroxyundecyl)-1-methylthymine                     ##STR32##                                                                    26     3-(6-Propylamino-5-hydroxyhexyl)-1-methylthymine                        ##STR33##                                                                    27     1-(8-hydroxy-9-(N-benzyl)aminononyl)-3,7-dimethylxanthine               ##STR34##                                                                    28     1-(5-hydroxy-6-(N-octyl)aminohexyl)-3,7-dimethylxanthine                ##STR35##                                                                    29     1-(5-hydroxy-6-(N-(4-phenyl)butyl)aminohexyl)-3,7-dimethylxanthine            .                                                                       ##STR36##                                                                    30     1-(6-Undecylamino-5-hydroxyhexyl)-3,7-dimethylxanthine                  ##STR37##                                                                    31     1-(5-hydroxy-6-(N-cyclohexylmethyl)aminohexyl)-3,7-dimethylxanthine           8                                                                       ##STR38##                                                                    32     1-(5-hydroxy-6-(N-(6-hydroxy)hexyl)aminohexyl)-3,7-dimethylxanthine           2                                                                       ##STR39##                                                                    33     1-(5-hydroxy-6-(N,N-dihexyl)aminohexyl)-3,7-dimethylxanthine            ##STR40##                                                                    34     1-(5-hydroxy-6-(N-(4-methoxy)benzyl)aminohexyl)-3,7-dimethylxanthin           e                                                                       ##STR41##                                                                    35     1-(8-hydroxy-9-(N-octyl)aminononyl)-3,7-dimethylxanthine                ##STR42##                                                                    36     1-(5-hydroxy-6-(N-tetradecyl)aminohexyl)-3,7-dimethylxanthine           ##STR43##                                                                    37     1 6-(Cyclopropylmethylamino)-5-hydroxyhexyl)!-3,7-dimethylxanthine            O                                                                       ##STR44##                                                                    38     1-(6-Decylamino-5-hydroxyhexyl)-3,7-dimethylxanthine                    ##STR45##                                                                    39     1-(6-Dodecylamino-5-hydroxyhexyl)-3,7-dimethylxanthine                  ##STR46##                                                                    40     1-(11-Benzylamino-10-hydroxyundecyl-3,7-dimethylxanthine                ##STR47##                                                                    41     1-(9-Decylamino-8-hydroxynonyl)-3,7-dimethylxanthine                    ##STR48##                                                                    42     1-(9-Dodecylamino-8-hydrononyl)-3,7-dimethylxanthine                    ##STR49##                                                                    43     1-(9-Tetradecylamino-8-hydroxynonyl)-3,7-dimethylxanthine               ##STR50##                                                                    44     1-(11-Hexylamino-10-hydroxyundecyl)-3,7-dimethylxanthine                ##STR51##                                                                    45     1-(11-Octylamino-10-hydroxyundecyl-3,7-dimethylxanthine                 ##STR52##                                                                    46     1-(6-Allylamino-5-hydroxyhexyl)-3,7-dimethylxanthine                    ##STR53##                                                                    47     1-(11-Allylamino-10-hydroxyundecyl)-3,7-dimethylxanthine                ##STR54##                                                                    48     1-(6-N-Methyloctadecylamino-5-hydroxyhexyl)-3,7-dimethylxanthine        ##STR55##                                                                    49     1-(11-Decylamino-10-hydroxyundecyl)-3,7-dimethylxanthine                ##STR56##                                                                    50     1-(11-Dodecylamino-10-hydroxyundecyl)-3,7-dmethylxanthine               ##STR57##                                                                    51     1-(11-Tetradecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine           ##STR58##                                                                    52     1- 11-(4-Fluorobenzylamino)-10-hydroxyundecyl!-3,7-dimethylxanthine           N                                                                       ##STR59##                                                                    53     1- 11-(4-Trifluoromethylbenzylamino)-10-hydroxyundecyl!3,7-dimethyl           xanthine                                                                ##STR60##                                                                    54     1- 11-(3-Diethylaminopropylamino)-10-hydroxyundecyl!-3,7-dimethylxa           nthine                                                                  ##STR61##                                                                    55     N,N'-bis (10-yl-9-hydroxydecyl)-3,7-dimethylxanthine!diaminododecan           e                                                                       ##STR62##                                                                    56     1-(14-Bromo-13-hydroxytetradecyl)-3,7-dimethylxanthine                  ##STR63##                                                                    57     1- 11-(4-Aminobenzylamino)-10-hydroxyundecyl!-3,7-dimethylxanthine            N                                                                       ##STR64##                                                                    58     1- 11-(3,4,5-Trimethoxybenzylamino)-10-hydroxyundecyl!-3,7-dimethyl           xanthine                                                                ##STR65##                                                                    59     1- 11-(3-Butoxypropylamino)10-hydroxyundecyl}3,7-dimethylxanthine       ##STR66##                                                                    60     1-(14-Octylamino-13-hydroxytetradecyl)-3,7-dimethylxanthine             ##STR67##                                                                    61     1-(11-Propylamino-10-hydroxyundecyl)-3,7-dimethylanthine                ##STR68##                                                                    62     1-(11-Undecylamino-10-hydroxyundecyl-3,7-dimethylxanthine               ##STR69##                                                                    63     1-(11-Phenylamino-10-hydroxyundecyl)-3,7-dimethylxanthine               ##STR70##                                                                    64     N,N-bis 11-yl-10-hydroxyundecyl)-3,7-dimethylxanthine!undecylamine            N                                                                       ##STR71##                                                                    65     1-(11-Octadecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine            ##STR72##                                                                    66     1- 9-(N-Methyloctylamino-8-hydroxynonyl)!-3,7-dimethylxanthine          ##STR73##                                                                    67     1-(4-Tetradecylamino-3-hydroxybutyl)-3,7-dimethylxanthine               ##STR74##                                                                    68     1- 9-(2-hydroxydecyl-1-amino)nonyl!-3,7-dimethylxanthine                ##STR75##                                                                    69     1-(6-Octadecylamino-5-hydroxyhexyl)-3,7-dimethylxanthine                ##STR76##                                                                    70     1- 11-(N-Octylacetamido)10-hydroxyundecyl!-3,7-dimethylxanthine         ##STR77##                                                                    71     2-(11-Octylamino-10-hydroxyundecyl)-N-methylbenzamide                   ##STR78##                                                                    72     1-(11-(N-Methyl-N-octylamino)-10-hydroxyundecyl)-3,7-dimethylxanthi           ne                                                                      ##STR79##                                                                    73     N-(11-Octylamino-10-hydroxyundecyl)piperidine                           ##STR80##                                                                    74     2-(11-Octylamino-10-hydroxyundecyl)-1,3-dihydroxybenzene                ##STR81##                                                                    75     1- 11-Amino-10-hydroxyundecyl)-3,7-dimethylxanthine                     ##STR82##                                                                    76     1-(11-Hexadecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine            ##STR83##                                                                    77     1-(11-Tridecylamino-10-hydroxylundecyl)-3,7-dimethylxanthine            ##STR84##                                                                    78     1- 11-Dihexylamino-10-hydroxyundecyl)-3,7-dimethylxanthine              ##STR85##                                                                    79     1-(11-Pentadecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine           ##STR86##                                                                    80     1- 11-(N,N-Diethanolamino)undecyl!-3,7-dimethylxanthine                 ##STR87##                                                                    81     1- 11-(2-Piperidinoethylamino)-10-hydroxyundecyl!-3,7-dimethylxanth           ine                                                                     ##STR88##                                                                    82     1- 11-(4-Methyl-1-yl-piperazino)-10-hydroxyundecyl!-3,7-dimethyxant           hine                                                                    ##STR89##                                                                    83     1- 11-Hydroxy-10-aminoundecyl!-3,7-dimethylxanthine                     ##STR90##                                                                    84     1- 11-(4-Chlorobenzyl)-10-hydroxyundecyl!-3,7-dimethylxanthine          ##STR91##                                                                    85     1- 11-(2,4,6-Trimethoxybenzylamino)-10-hydroxyundecyl!-3,7-dimethyl           xanthine                                                                ##STR92##                                                                    86     1-(11-tert-Butylamino-10-hydroxyundecyl)-3,7-dimethylxanthine           ##STR93##                                                                    87     6-(11-dodecylamino-10-hydroxyundecoxy)-2-hydroxy-3,7-methylpurine       ##STR94##                                                                    88     N,N-bis- (11-yl-10-hydroxyundecyl)-3,7-dimethylxanthine!dodecylamin           e                                                                       ##STR95##                                                                    89     1- 11-(3,4,5-Trimethoxyphenylamino)-10-hydroxyundecyl!-3,7-dimethyl           xanthine                                                                ##STR96##                                                                    90     1- 11-(N-Methyl-N-dodecylamino)-10-hydroxyundecyl!-3,7-dimethylxant           hine                                                                    ##STR97##                                                                    91     1- 11-(N-Dodecylacetamido)-10-hydroxyundecyl!-3,7-dimethylxanthine            O                                                                       ##STR98##                                                                    92     1- 11-(N-Tetradecylacetamido)-10-hydroxyundecyl!-3,7-dimethylxanthi           ne                                                                      ##STR99##                                                                    93     1- 11-(3,4,5-Trimethoxybenzylacetamido)-10-hydroxyundecyl!-3,7-dime           thylxanthine                                                            ##STR100##                                                                   94     1- 11-(N-Dodecylacetamido)-10-acetoxyundecyl!-3,7-dimethylxanthine      ##STR101##                                                                   95     1- 11-(N-Methyl-N-dodecylamino)-10-acetoxyundecyl!-3,7-dimethylxant           hine                                                                    ##STR102##                                                                   96     1- 11-(Morpholine-4-yl)-10-hydroxyundecyl!-3,7-dimethylxanthine         ##STR103##                                                                   97     1- 11-(Dodecyl benzamido)-10-hydroxyundecyl!-3,7-dimethylxanthine       ##STR104##                                                                   98     1- 11-(3,5-Dimethoxybenzylamino)-10-hydroxyundecyl!-3,7-dimethylxan           thine                                                                   ##STR105##                                                                   99     1- 7-(3-Octyl-2-oxo-5-oxazolidinyl)heptyl!-3,7-dimethylxanthine         ##STR106##                                                                   100    1- 9-(N-Dodecyl-2-oxazolidin-5-yl)nonyl!-3,7-dimethylxanthine           ##STR107##                                                                   __________________________________________________________________________

Pharmaceutical Formulations

A suitable formulation will depend on the nature of the disorder to betreated, the nature of the medicament chosen, and the judgment of theattending physician. In general, the inventive compounds are formulatedeither for injection or oral administration, although other modes ofadministration such as transmucosal or transdermal routes may beemployed. Suitable formulations for these compounds can be found, forexample, in Remington's Pharmaceutical Sciences (latest edition), MackPublishing Company, Easton, Pa.

The inventive compounds and their pharmaceutically acceptable salts canbe employed in a wide variety of pharmaceutical forms. The preparationof a pharmaceutically acceptable salt will be determined by the chemicalnature of the compound itself, and can be prepared by conventionaltechniques readily available. Thus, if a solid carrier is used, thepreparation can be tableted, placed in a hard gelatin capsule in powderor pellet form or in the form of a troche or lozenge. The amount ofsolid carrier will vary widely but preferably will be from about 25 mgto about 1 gram, wherein the amount of inventive compound per dose willvary from about 25 mg to about 1 gram for an adult. When a liquidcarrier is used, the preparation will be in the form of a syrup,emulsion, soft gelatin capsule, sterile injectable liquid such as anampule or nonaqueous liquid suspension. Where the inventive compositionis in the form of a capsule, any routine encapsulation is suitable, forexample, using the aforementioned carriers in a hard gelatin capsuleshell. Where the composition is in the form of a soft gelatin shellcapsule, any pharmaceutical carrier routinely used for preparingdispersions of suspensions may be considered, for example, aqueous gums,celluloses, silicates or oils and are incorporated in a soft gelatincapsule shell. A syrup formulation will generally consist of asuspension or solution of the compound or salt thereof in a liquidcarrier (e.g., ethanol, polyethylene glycol, coconut oil, glycerine orwater) with a flavor or coloring agent.

The amount of inventive compound required for therapeutic effect ontopical administration will, of course, vary with the compound chosen,the nature and severity of the disease and the discretion of thetreatment provider. Parenteral includes intravenous, intramuscular,subcutaneous, intranasal, intrarectal, intravaginal or intraperitonealadministration. Appropriate dosage forms for such administration may beprepared by conventional techniques. A typical parenteral compositionconsists of a solution or suspension of the inventive compound or a saltthereof in a sterile or non-aqueous carrier, optionally containing aparenterally acceptable oil, for example polyethylene glycol,polyvinylpyrrolidone, lecithin, arachis oil, or sesame oil. The dailydosage for treatment of sepsis or another severe inflammatory conditionvia parenteral administration is suitable from about 0.001 mg/kg toabout 40 mg/kg, preferably from about 0.01 mg/kg to about 20 mg/kg of aninventive compound or a pharmaceutically acceptable salt thereofcalculated as the free base.

The inventive compounds may be administered orally. The daily dosageregimen for oral administration is suitably from about 0.1 mg/kg toabout 1000 mg/kg per day. For administration the dosage is suitably fromabout 0.001 mg/kg to about 40 mg/kg of the inventive compound or apharmaceutically acceptable salt thereof, calculated as the free base.The active ingredient may be administered from 1 to 6 times a day,sufficient to exhibit activity.

The inventive compounds may be administered by inhalation (e.g.,intranasal or oral). Appropriate dosage forms include an aerosol or ametered dose inhaler, as prepared by conventional techniques. The dailydosage is suitably from about 0.001 mg/kg to about 40 mg/kg of theinventive compound or a pharmaceutically acceptable salt thereof,calculated as the free base. Typical compounds for inhalation are in theform of a solution, suspension or emulsion that may be administered as adry powder or in the form of an aerosol using a conventional propellant.

The following examples are illustrative of specific, preferredembodiments of the invention; however, these examples are not intendedto be construed as limiting the scope of the invention as disclosedherein.

EXAMPLE 1

This example illustrates the synthesis of several compounds that areused as intermediates for the synthesis of other compounds.

1-(8,9-Oxidononyl)-3,7-dimethylxanthine was synthesized as follows: Amixture of theobromine (17.64 g, 98 mmol) and sodium hydride (2.35 g, 98mmol) in dimethylsulfoxide (250 ml) was stirred for 15 minutes.9-Bromo-1-nonene (20.0 g, 98 mmol) was added and stirring continued for3 days. The reaction mixture was poured into water (300 mi) andextracted with dichloromethane (4×200 ml). The combined organic layerswere washed with saturated aqueous sodium chloride solution (2×150 ml),dried over sodium sulfate, and the solvents evaporated under vacuum. Theresidue was crystallized (dichloromethane-ether) to give1-(8-nonenyl)-3,7-dimethylxanthine (24.34 g, 99% yield) as whitecrystals.

A solution of 1-(8-nonenyl)-3,7-dimethylxanthine (810 mg, 2.7 mmol),4-methylmorpholine-N-oxide (340 mg, 2.9 mmol) and 2.5% osmium tetroxidein t-butanol (3 drops) in acetone (20 ml) and water (20 ml) was stirredfor 24 hours. Saturated aqueous sodium dithionite solution (5 ml) wasadded. After stirring for 15 minutes, the reaction was extracted with25% ethanol-dichloromethane (4×50 ml). The combined organic phases weredried over sodium sulfate and the solvents evaporated under vacuum. Thesolid residue was recrystallized (ethanol-chloroform) to give1-(8,9-dihydroxynonyl)-3,7-dimethylxanthine (490 mg, 54% yield).

A mixture of 1-(8,9-dihydroxynonyl)-3,7-dimethylxanthine (428 mg, 1.3mmol) and 30% hydrogen bromide in acetic acid (0.8 ml, 3.9 mmol) wasstirred for 90 minutes. The solution was poured into a mixture of water(10 ml), sodium bicarbonate (1.35 g), and dichloromethane (10 ml). After10 minutes of vigorous stirring the layers were separated and theaqueous portion was extracted with dichloromethane (3×15 ml). Thecombined organic phases were dried over sodium sulfate and the solventwas evaporated under vacuum to give1-(8-acetoxy-9-bromononyl)-3,7-dimethylxanthine (550 mg, 96% yield) as ayellow oil. Without further purification, the oil was dissolved inmethanol (5 ml) and then a 1M solution of sodium methoxide in methanol(1.4 ml) was added. After 30 minutes the reaction mixture was pouredinto water (30 ml) and extracted with dichloromethane (3×40 ml). Thecombined organic phases were dried over sodium sulfate and the solventsevaporated under vacuum. The solid residue was recrystallized(dichloromethane-petroleum ether) to give1-(8,9-oxidononyl)-3,7-dimethylxanthine (380 mg, 91% yield).

1-(5,6-Oxidohexyl)-3,7-dimethylxanthine was synthesized as follows: Amixture of 1-bromohexene (10.7 g, 66 mmol), sodium hydride (1.58 g, 66mmol), and theobromine (11.9 g, 66 mmol) in dimethylsulfoxide (100 ml)was stirred for 43 hours. The solution was treated with water (200 ml)and then extracted with dichloromethane (3×80 ml). The combined extractswere washed with water (3×100 ml), dried over magnesium sulfate, andthen the solvent was evaporated under vacuum to give1-(5-hexenyl)-3,7-dimethylxanthine (17 g, 98% yield) as a white powder.

To 1-(5-hexenyl)-3,7-dimethylxanthine (1.07 g, 4.1 mmol) and4-methylmorpholine-N-oxide (1.44 g, 12.3 mmol) in water (20 ml) andacetone (10 ml) was added 2.5% solution of osmium tetraoxide int-butanol (6 drops). After stirring for 48 hours, the mixture wastreated with 20% aqueous sodium dithionite solution (20 ml). After 2minutes, the mixture was extracted with 25% ethanol-dichloromethane(3×30 ml). The combined extracts were dried over magnesium sulfate andthe solvents were evaporated under vacuum to give1-(5,6-dihydroxyhexyl)-3,7-dimethylxanthine (750 mg, 62% yield) as awhite powder.

To 1-(5,6-dihydroxyhexyl)-3,7-dimethylxanthine (1.0 g, 3.38 mmol) wasadded 30% hydrogen bromide-acetic acid (3.4 ml) over 30 seconds and thenstirred until all of the solid had dissolved (2.5 hours). The solutionwas poured carefully over a mixture of sodium bicarbonate (12 g) and icewater (50 ml). After carbon dioxide evolution had subsided, the mixturewas extracted with dichloromethane (3×25 ml). The combined extracts weredried over magnesium sulfate and the solvent was evaporated under vacuumto give 1-(5-acetoxy-6-bromohexyl)-3,7-dimethylxanthine (1.3 g, 96%yield) as a viscous oil which was dissolved in methanol (5 mL). A 1Msolution of sodium methoxide in methanol (3.9 ml) was added over 30seconds. After stirring for 20 minutes, the solution was treated withwater (20 ml) and then extracted with dichloromethane (3×15 ml). Thecombined extracts were dried over magnesium sulfate and the solventswere evaporated under vacuum to give1-(5,6-oxidohexyl)-3,7-dimethylxanthine (900 mg, 100% yield) as whitecrystals.

3-(5,6-Oxidohexyl)-1-methyluracil was synthesized as follows: A mixtureof sodium hydride (86 mg, 3.6 mmol) and 1-methyluracil (500 mg, 4 mmol)in dimethyl sulfoxide (25 ml) was stirred for 15 minutes, and then6-bromo-1-hexene (647 mg, 4 mmol) was added. After stirring for 20hours, the reaction mixture was poured into water (50 ml) and extractedwith 20% ethanol-dichloromethane (3×50 ml). The combined organic layerswere washed with saturated aqueous sodium chloride solution (20 ml) anddried over sodium sulfate. The solvent was evaporated under vacuum togive a residue which was purified by column chromatography (silica,ethyl acetate) to give 3-(5-hexenyl)-1-methyluracil (598 mg, 72% yield).

A solution of 3-(5-hexenyl)-1-methyluracil (598 mg, 2.9 mmol),4-methylmorpholine-N oxide (408 mg, 3.5 mmol), and a 2.5% solution ofosmium tetroxide in t-butanol (3 drops) in acetone (15 ml) and water (5ml) was stirred for 3 days. Saturated aqueous sodium hydrosulfitesolution (10 ml) was added and the mixture was stirred for 15 minutes.Water (50 ml) was added and the mixture was extracted with 20%ethanol-dichloromethane (4×40 ml). The combined organic layers weredried over sodium sulfate and the solvents were evaporated under vacuumto give 3-(5,6-dihydroxyhexyl)-1-methyluracil (461 mg, 66% yield) as acolorless oil.

3-(5,6-Dihydroxyhexyl)-1-methyluracil (350 mg, 1.4 mmol) was stirredwith 30% hydrogen bromide in acetic acid (0.87 ml, 4.3 mmol) for 45minutes. The solution was added to a mixture of sodium bicarbonate (1.6g), water (10 ml) and dichloromethane (20 ml). After 15 minutes ofvigorous stirring, the layers were separated and the aqueous layer wasextracted with dichloromethane (3×40 ml). The combined organic layerswere dried over sodium sulfate and the solvent was evaporated undervacuum to give 3-(5-acetoxy-6-bromohexyl)-1-methyluracil (500 mg, 100%yield). The bromoacetate thus obtained was used in the next step withoutfurther purification. 3-(5-Acetoxy-6-bromohexyl)-l-methyluracil (360 mg,1.0 mmol) was dissolved in methanol (5 ml) and treated with a solutionof 1M sodium methoxide in methanol (1 ml). After stirring for 15minutes, the solution was poured into water (10 ml) and extracted withdichloromethane (3×30 ml). The combined organic layers were dried oversodium sulfate and the solvents were evaporated to give3-(5,6-oxidohexyl)-1-methyluracil (150 mg, 67% yield) as a colorlessoil.

3-(5,6-Oxidohexyl)-1-methylthymine was synthesized as follows: A mixtureof sodium hydride (343 mg, 14 mmol) and 1-methylthymine (2.00 g, 14mmol) in dimethylsulfoxide (30 ml) was stirred for 15 minutes, and then6-bromo-1-hexene (2.30 g, 14 mmol) was added. After stirring for 69hours, the reaction mixture was poured into water (100 ml) and extractedwith dichloromethane (4×50 ml). The combined organic layers were washedwith saturated aqueous sodium chloride solution (40 ml), dried oversodium sulfate, and then the solvent was evaporated under vacuum to givea residue which was recrystallized (dichloromethane-ethyl ether) to give3-(5-hexenyl)-1-methylthymine (2.80 g, 88% yield).

A solution of 3-(5-hexenyl)-1-methylthymine (2.00 g, 9 mmol),4-methylmorpholine-N-oxide (1.17 mg, 10 mmol), and osmium tetroxide(0.15 ml of a 2.5% solution in t-butanol) in acetone (15 ml) and water(10 ml) was stirred for 20 hours. Saturated aqueous sodium hydrosulfitesolution (10 ml) was added and after 15 minutes of stirring, the mixturewas extracted with 20% ethanol-dichloromethane (4×40 ml). The combinedorganic layers were dried over sodium sulfate and the solvents wereevaporated under vacuum to give a solid residue. The solid wasrecrystallized (ethanol) to give 3-(5,6-dihydroxyhexyl)-1-methylthymine(2.00 g, 89% yield).

3-(5,6-Dihydroxyhexyl)-1-methylthymine (1.65 g, 6.4 mmol) was stirredwith 30% hydrogen bromide in acetic acid (3.8 ml, 19.3 mmol) for 1.5hours. The mixture was then added to a mixture of sodium bicarbonate(6.7 g), water (40 ml), and dichloromethane (50 ml). After 15 minutes ofvigorous stirring, the layers were separated and the aqueous layer wasextracted with dichloromethane (2×50 ml). The combined organic layerswere dried over sodium sulfate and the solvent was evaporated undervacuum to give 3-(5-acetoxy-6-bromohexyl)-1-methylthymine (2.30 g, 100%yield). The bromoacetate was used in the next step without furtherpurification. 3-(5-Acetoxy-6-bromohexyl)-1-methylthymine (2.30 g,6.4mmol) was dissolved in methanol (10 ml) and a solution of 1M sodiummethoxide in methanol (7 ml) was added. After stirring for 15 minutes,the solution was poured into water (60 ml) and extracted with 20%ethanol-dichloromethane (2×70 ml). The combined organic layers weredried over sodium sulfate and the solvents were evaporated under vacuumto give 3-(5,6-oxidohexyl)-1-methylthymine (1.30 g, 85% yield) as awhite solid.

3-(5,6-Oxidohexyl)-1-methylbenzoyleneurea was synthesized as follows: Asolution of sodium hydride (0.76 g, 30 mmol) and benzoyleneurea (4.86 g,30 mmol) in dimethylsulfoxide (100 ml) was stirred for 10 minutes andthen methyl iodide (1.87 ml, 30 mmol) was added. After stirring for 14hours, water (100 ml) was added and the solution was extracted withdichloromethane (3×100 ml). The mixture was filtered and thedichloromethane phase was dried over sodium sulfate. After evaporationof the solvent under vacuum, the residue was recrystallized(dichloromethane) to give 1-methylbenzoyleneurea (1.3 g, 25% yield) as awhite solid.

A solution of sodium hydride (0.17 g, 6.8 mmol) and1-methylbenzoyleneurea (1.07 g, 6.1 mmol) in dimethyl sulfoxide (50 ml)was stirred for 10 minutes and then 1-bromohexene (0.82 ml, 6.8 mmol)was added. After 14 hours, water (50 ml) was added and the solution wasextracted with dichloromethane (3×30 ml). The combined organic phaseswere washed with water (3×50 ml), dried over sodium sulfate, and thesolvent was evaporated under vacuum to give3-(5-hexenyl)-1-methylbenzoyleneurea (1.51 g, 96%) as a white solid.

A solution of 3-(5-hexenyl)-1-methylbenzoyleneurea (1.5 g, 5.8 mmol),4-methylmorphline-N-oxide (0.87 g, 7.4 mmol), and potassium osmate(IV)dihydrate (0.021 g, 0.1 mmol) in acetone (12.5 ml) and water (4 ml) wasstirred. After 18 hours, a 20% aqueous solution hydrosulfite (20 ml) wasadded and stirred for 30 minutes. The solution was extracted withdichloromethane (3×75 ml). The combined organic phases were dried oversodium sulfate and the solvent was evaporated under vacuum. The residuewas purified by flash chromatography (silica, 5%methanol-dichloromethane) to give3-(5,6-dihydroxyhexyl)-1-methylbenzoyleneurea (1.59 g, 94%) as a whitesolid.

A mixture of 3-(5,6-dihydroxyhexyl)-1-methylbenzoyleneurea (0.92 g, 3.1mmol) in 30% hydrogen bromide in acetic acid (0.63 ml, 9.3 mmol) wasstirred for 90 minutes. The reaction mixture was poured into a mixtureof sodium bicarbonate (0.78 g, 9.3 mmol), water (20 ml), anddichloromethane (20 ml). The phases were separated and the aqueous phasewas extracted with dichloromethane (2×20 ml). The combined organicphases were washed with brine (20 ml), dried over sodium sulfate, andthe solvent was evaporated under vacuum to give3-(5-acetoxy-6-bromohexyl)-1-methylbenzoyleneurea (1.2 g, 96%).

To a 1M solution of sodium methoxide in methanol (3.1 ml) was added1-(5-acetoxy-6-bromohexyl)-3-methylbenzoyleneurea (1.17 g, 2.9 mmol) inmethanol (25 ml) over 5 minutes. After stirring for 1 hour, water (50ml) was added. The solution was extracted with dichloromethane (3×25ml). The combined organic phases were dried over sodium sulfate and thesolvents were evaporated under vacuum to give3-(5,6-oxidohexyl)-1-methylbenzoyleneurea (0.77 g, 97%) as a whitesolid.

1-(5,6-Oxidohexyl)glutarimide was synthesized as follows: A mixture ofglutarimide (2.00 g, 7.7 mmol) and sodium hydride (425 mg, 17.7 mmol) indimethyl sulfoxide (40 ml) was stirred for 20 minutes and then6-bromo-1-hexene (2.90 g, 17.7 mmol) was added. After 20 hours ofstirring, the reaction was poured into water (100 ml) and extracted withdichloromethane (4×50 ml). The combined organic layers were washed withwater (50 ml) and then with saturated aqueous sodium chloride solution(50 ml). After drying over sodium sulfate the solvent was evaporatedunder vacuum to give 1-(5-hexenyl)glutarimide (2.92 g, 85% yield).

To a solution of 1-(5-hexenyl)glutarimide (630 mg, 3.2 mmol) indichloromethane (10 ml) was added sodium bicarbonate (2.20 g, 26 mmol)in water (10 ml) by 50% m-chloroperoxybenzoic acid (2.5 g, 7.2 mmol).After stirring for 17 hours, sodium metabisulfite (1.7 g, 9.0 mmol) wasadded and stirred for 30 minutes. The mixture was extracted withdichloromethane (3×10 ml) and then the combined organic layers werewashed with saturated aqueous sodium bicarbonate solution (10 ml). Afterdrying over sodium sulfate and evaporation of the solvent under vacuum,the residue was purified by column chromatography (silica, 10%ethanol-dichloromethane) to give 1-(5,6-oxidohexyl)glutarimide (180 mg,27% yield).

EXAMPLE 2

This example illustrates a method for synthesis of1-(8-hydroxy-9-(N-benzyl)aminononyl)-3,7-dimethylxanthine (compound no.27). A mixture of 1-(8,9-oxidohexyl)-3,7-dimethylxanthine (500 mg, 1.6mmol) from Example 1 and benzylamine (2.0 g, 19 mmol) was heated at 150°C. for 4 hours. After cooling to ambient temperature, ether (30 ml) wasadded. The precipitate was washed with cold ether to give (compound no.27) (278 mg, 41% yield).

EXAMPLE 3

This example illustrates the synthesis of1-(5-hydroxy-6-(N-octyl)aminohexyl)-3,7-dimethylxanthine (compound no.28). A mixture of 1-(5,6-oxidohexyl)-3,7-dimethylxanthine (400 mg, 1.4mmol) synthesized in example 1, and 1-octylamine (391 mg, 3 mmol) washeated at 135° C. for 4 hours. After cooling to ambient temperature,ether (15 ml) was added. The precipitate was washed several times withhexane to give compound no. 28 (537 mg, 94% yield).

EXAMPLE 4

This example illustrates the synthesis of1-(5-hydroxy-6-(N-(4-phenyl)butyl)amino)hexyl)-3,7-dimethylxanthine(compound no. 29). A mixture of 1-(5,6-oxidohexyl)-3,7-dimethylxanthine(300 mg, 1,1 mmol) from example 1 and 4-phenyl-1-butylamine (322 mg, 2.2mmol) was heated at 130° C. for 70 minutes. After cooling to ambienttemperature, the residue was dissolved in dichloromethane (2 ml) andadded to ether (20 ml). The precipitate was washed several times withhexane to give compound no. 29 (280 mg, 60% yield).

EXAMPLE 5

This example illustrates the synthesis of1-(5-hydroxy-6-(N-undecyl)aminohexyl)-3,7-dimethylxanthine (compound no.30). A mixture of 1-(5,6-oxidohexyl)-3,7-dimethylxanthine (300 mg, 1.1mmol) from example 1 and 1-undecylamine (754 mg, 4.4 mmol) was heated at100° C. for 4 hours and then at 130° C. for 1 hour. After cooling toambient temperature, ether (10 ml) was added. The waxy precipitate waswashed several times with hexane to give compound no. 30 (403 mg, 82%yield).

EXAMPLE 6

This example illustrates the synthesis of1-(5-hydroxy-6-(N-cyclohexylmethyl)aminohexyl)-3,7-dimethylxanthine(compound no. 31). A mixture of 1-(5,6-oxidohexyl)-3,7-dimethylxanthine(300 mg, 1.1 mmol) from example 1 and cyclohexanemethylamine (249 mg,2.2 mmol) was heated at 100° C. for 5 hours and then at 120° C. for 1hour. After cooling to ambient temperature, ether (7 ml) and hexane (10ml) were added. The precipitate was washed several times with hexane togive compound no. 31 (294 mg, 68% yield).

EXAMPLE 7

This example illustrates the synthesis of1-(5-hydroxy-6-(N-(6-hydroxy)hexyl)aminohexyl)-3,7-dimethylxanthine(compound no. 32). A mixture of 1-(5,6-oxidohexyl)-3,7-dimethylxanthine(300 mg, 1.1 mmol) from example 1 and 6-amino-1-hexanol (754 mg, 2.6mmol) was heated at 120° C. for 2 hours. After cooling to ambienttemperature, ether (20 ml) was added. The precipitate was washed severaltimes with hexane to give compound no. 32 (321 mg, 74% yield).

EXAMPLE 8

This example illustrates the synthesis of1-(5-hydroxy-6-(N,N-dihexyl)aminohexyl)-3,7-dimethylxanthine (compoundno. 33). A mixture of 1-(5,6-oxidohexyl)3,7-dimethylxanthine (300 mg,1.1 mmol) from example 1 and dihexylamine (556 mg, 3.0 mmol) was heatedat 135° C. for 5 hours and then at 170° C. for 2 hours. After cooling toambient temperature, petroleum ether (20 ml) was added. After cooling ina freezer, the precipitate was washed several times with petroleum etherto give compound no. 33 (263 mg, 52% yield).

EXAMPLE 9

This example illustrates the synthesis of1-(5-hydroxy-6-(N-(4-methoxy)benzyl)aminohexyl)-3,7-dimethylxanthine(compound no. 34). A mixture of 1-(5,6-oxidohexyl)-3,7-dimethylxanthine(300 mg, 1.1 mmol) from example 1 and 4-methoxybenzylamine (0.7 g, 5mmol) was heated at 100° C. for 4 hours. After cooling to ambienttemperature, ether (10 ml) was added. The precipitate was washed severaltimes with petroleum ether to give compound no. 34 (355 mg, 78% yield).

EXAMPLE 10

This example illustrates the synthesis of3-(5-hydroxy-6-(N-propyl)aminohexyl)-1-methyuracil (compound no. 19). Amixture of 3-(5,6-oxidohexyl)-1-methyluracil (100 mg, 0.4 mmol) fromexample 1 and n-propylamine (10 ml) was heated in a sealed pressurebottle at 80°-90° C. for 69 hours. Evaporation of the unreactedn-propylamine gave a yellow oil which was crystallized(ether-dichloromethane) to give compound no. 19 (80 mg, 71%) as a whitesolid.

EXAMPLE 11

This example illustrates the synthesis of3-(5-hydroxy-6-(N-benzyl)aminohexyl)-1-methylbenzoyleneurea (compoundno.3). A mixture of 3-(5,6-oxidohexyl)-1-methylbenzoyleneurea (0.1 g,0.4 mmol) from example 1 and benzylamine (0.13 g, 1.2 mmol) was stirredunder argon at 115° C. After 3 hours, the unreacted benzylamine wasevaporated under vacuum. The residue crystallized on standing to give(compound no. 3) (0.14 g, 93% yield) as a white solid.

EXAMPLE 12

This example illustrates the synthesis of1-(5-hydroxy-6-(N-propyl)aminohexyl)-3,7-dimethylxanthine (compound no.14). A solution of 1-(5,6-oxohexyl)-3,7-dimethylxanthine (238 mg, 0.86mmol) from example 1 in n-propylamine (5 ml) was heated at 100° C. in asealed pressure bottle for 23 hours. After cooling to 4° C., the bottlewas unsealed and unreacted n-propylamine was evaporated under vacuum togive (compound no. 14) (190 mg, 64% yield) as a viscous oil.

EXAMPLE 13

This example illustrates the synthesis of1-(5-hydroxy-6-(N-benzyl)aminohexyl)-3,7-dimethylxanthine (compound no.13). A mixture of (5,6-oxidohexyl)-3,7-dimethylxanthine (500 mg, 1.8mmol) from example I and benzylamine (1.7 g, 15.8 mmol) was heated at150° C. for 4 hours. After cooling to ambient temperature, ether wasadded (20 ml). The precipitate was washed with cold ether to givecompound no. 13 (470 mg, 70% yield).

EXAMPLE 14

This example illustrates the synthesis of N² -(5-hydroxy-6-(N³-propyl)aminohexyl)-(N¹ -propyl)glutaric acid (compound no. 5). Asolution of 1-(5,6-oxidohexyl)glutarimide (60 mg, 0.3 mmol) from example1 in n-propylamine (5 ml) was heated in a sealed pressure bottle at80°-90° C. for 30 hours. Unreacted n-propylamine was evaporated undervacuum to give a residue which was triturated with ether to givecompound no. 5 (100 mg, 100% yield).

EXAMPLE 15

This example illustrates the synthesis of3-(5-hydroxy-6-(N-undecyl)aminohexyl)-1-methylthymine (compound no. 24).A mixture of 3-(5,6-oxidohexyl)-1-methylthymine from example 1 (250 mg,1.1 mmol) and 1-undecylamine (0.7 ml) were heated at 110° C. for 4hours. After cooling to ambient temperature, ether (5 ml) and petroleumether (10 ml) were added. After cooling to -10° C. for 2 hours, theprecipitate was washed several times with petroleum ether to givecompound no. 24 (361 mg, 80% yield).

EXAMPLE 16

This example illustrates the synthesis of3-(6-propylamino-5-hydroxyhexyl)-1-methylthymine (compound no. 26). Asolution of 3-(5,6-oxidohexyl)-1-methylthymine (200 mg, 0.8 mmol) fromexample 1 in n-propylamine (10 ml) was heated in a sealed pressurebottle at 100°-105° C. for 24 hours. After evaporation of unreactedn-propylamine, the residue was crystallized (ether) to give compound no.26 (162 mg, 68% yield).

EXAMPLE 17

This example illustrates the synthesis of1-(8-hydroxy-9-(N-octyl)aminononyl)-3,7-dimethylxanthine (compound no.35). A mixture of 1-(8,9-oxidohexyl)-3,7-dimethylxanthine (300 mg, 0.9mmol) from example 1 and octylamine (1 ml) were heated at 110° C. for 3hours. After cooling to room temperature, ether (10 ml) was added. Theprecipitate was washed several times with petroleum ether to givecompound no. 35 (342 mg, 85% yield).

EXAMPLE 18

This example illustrates the synthesis of1-(5-hydroxy-6-(N-tetradecyl)aminohexyl)-3,7-dimethylxanthine (compoundno. 36). A mixture of 1-(5,6-oxohexyl)-3,7-dimethylxanthine (300 mg, 1.1mmol) from example 1 and 1-tetradecylamine (604 mg, 2.8 mmol) was heatedat 110° C. for 3 hours. After cooling to ambient temperature, ether (6ml) was added. The precipitate was washed several times with petroleumether to give compound no. 36 (356 mg, 66% yield).

EXAMPLE 19

This example illustrates a method of synthesis for1-(9-tetradecylamino-8-hydroxynonyl)-3,7-dimethylxanthine (compound no.43). A mixture of 1-(8,9-oxidononyl)-3,7-dimethylxanthine (synthesizedin example 1 above, 1.00 g, 3.1 mmol) and anhydrous lithium perchlorate(329 mg, 3.1 mmol) was stirred in anhydrous acetonitrile (30 ml). Afteraddition of 1-tetradecylamine (Aldrich, 722 mg, 3.4 mmol), the mixturewas stirred at 60° C. for 4 hours. After cooling, water (50 ml) wasadded and the mixture was extracted with dichloromethane (3×50 ml). Thecombined organic layers were washed with water (30 ml) and saturatedaqueous salt solution (30 ml) and subsequently dried over sodiumsulfate. The solvent was removed under vacuum to give a white residue.Chromatography (neutral activity II alumina, dichloromethan/5%methanol)of the white residue resulted in 860 mg of compound no. 43 (52% yield).

EXAMPLE 20

This example illustrates a method of synthesis for compound no. 63.Sodium hydride (95%) (1.26 g, 50 mmol) was added to a solution oftheobromine (7.2 g, 40 mmol) in dimethylsulfoxide (300 ml). After 20minutes of stirring, undecenylmesylate (7.95 g, 30 mmol) was added andstirred for 12 hours at room temperature. The reaction, warmed to70°-80° C., was stirred for 4 hours. The reaction mixture was pouredinto a separatory funnel containing 1 L of water and extracted withdichloromethane (5×200 ml). The organic extracts were combined, washedwith water (100 ml) and brine (100 ml), dried over anhydrous magnesiumsulfate and concentrated under reduced pressure. A crude productobtained was further purified by flash chromatography over silica gelusing an eluant of 20% hexane/dichloromethane to obtain 4.6 g of1-10-undecenyl)-3,7-dimethylxanthine (yield, 46.3%).

A solution of 1-(10-undecenyl)-3,7-dimethylxanthine, prepared above (4.3g, 13 mmol), 4-methylmorpholine-N-oxide (1.942 g, 16.6 mmol) andpotassium osmate dihydrate (9.5 mg; 0.026 mmol) in acetone (45 ml) andwater (10 ml) was stirred for 6 hours. A solution of 20% aqueous sodiumsulphite (12 ml) was added and stirred for 30 minutes. The reactionmixture was extracted with 25% ethanol/dichloromethane (4×100 ml). Thecombined organic extracts were dried over anhydrous magnesium sulfate,concentrated under reduced pressure and purified by flash chromatographyover silica gel using a methanol/5% dichloromethane eluent to obtain 3.6g of 1-(10, 11-dihydroxyundecanyl)-3,7-dimethylxanthine (yield, 76%).

1-(10,11-dihydroxyundecanyl)-3,7-dimethylxanthine, prepared above (3.6g, 10 mmol), was stirred with hydrogen bromide (6.2 ml, 8.4 g of a 30%solution in acetic acid, 31.1 mmol) for 90 minutes. The mixture was thenadded to a flask containing 100 ml aqueous sodium bicarbonate solutionand 75 ml dichloromethane. After 10 minutes of vigorous stirring, thelayers were separated and an aqueous portion washed with dichloromethane(3×75 ml). The organic portions were combined, dried over magnesiumsulfate, and evaporated to give1-(10-acetoxy-11-bromoundecanyl)-3,7-dimethylxanthine (3.6 g). Withoutfurther purification, the bromoacetate was taken up in methanol (25 ml)and treated with a solution of sodium methoxide (prepared from 0.28 g,12.2 mmol sodium, and 25 ml methanol). After 30 minutes, most of thesolvent was removed under reduced pressure and the residue was extractedwith dichloromethane (3×75 ml). The organic portions were combined,dried over magnesium sulfate and concentrated under reduced pressure togive an off-white solid, purified by column chromatography over silicagel using dichloromethane/3% methanol eluant to obtain 2.0 g of1-(10,11-oxidoundecanyl)-3,7-dimethylxanthine (yield, 57.5%).

A mixture of 1-(10,11-oxidoundecyl)-3,7-dimethylxanthine, prepared above(500 mg, 1.4 mmol), and lithium perchlorate (from Aldrich, 149 mg, 1.4mmol) was stirred in anhydrous acetonitrile (from Aldrich, 20 ml) untilhomogeneous. Aniline (from Aldrich, 670 mg, 7.2 mmol) was added, and themixture stirred at ambient temperature for 16 hours, then at a refluxtemperature for 3 hours. The residue was directly deposited on a silicacolumn. Chromatography using a dichloromethane/10% methanol gradientproduced 0.45 g of compound no. 63 (73% yield).

EXAMPLE 21

This example illustrates data of proliferative activity of variouscompounds for inducing CMV promoter activity. The CMV promoter assaymeasures gene transcription and translation activity wherein any activecompounds will have cytotoxic activity for inhibiting cellular proteinsynthesis in transformed (adenovirus) cells. Each compound was testedand the data is listed in Table I below. Compound no. 30 was the mostcytotoxic compound tested.

                  TABLE I                                                         ______________________________________                                        Compound      IC.sub.50 (μM)                                               ______________________________________                                        13            >500                                                            28             50                                                             29            >100                                                            30             15                                                             31            >100                                                            32            >100                                                            33             100                                                             5            >500                                                            19            >500                                                            26            >500                                                            ______________________________________                                    

EXAMPLE 22

This example shows the effects of five compounds on inhibition of mastcell degranulation by a serotonin release assay. This assay provides anin vitro model for an allergy and asthma therapeutic product. Table IIbelow shows the results of five compounds (see above

                  TABLE II                                                        ______________________________________                                        Compound     % Inhibition                                                                            Concentration (μM)                                  ______________________________________                                        13           28%       100                                                    27           29%       50                                                     30           too toxic 50                                                     35           too toxic 50                                                     19           inactive  50                                                     ______________________________________                                    

EXAMPLE 23

This example illustrates dose response curves used to generate 50%inhibition concentration (IC₅₀) of for both cyclosporin A (CsA, FIG. 1A)and various compounds (FIG. 1B) for murine thymocyte proliferation,co-stimulated by Concanavalin A (ConA) and interleukin-2 (IL-2). Con A,used to activate CD3, induces T-cell proliferation and differentiation.Thymuses, obtained from normal, female Balb/C mice, were dissociated andplated into 96-well plates at a density of 2×10⁵ cells/well. ConA (0.25mg/ml) and IL-2 (15 U/ml) were added to the wells. The cells wereincubated for 4 days at 37° C. On day 4, the cells were pulsed withtritiated thymidine and incubated for an additional 4 hours. The amountof tritiated thymidine dye incorporated by the harvested cells wasdetermined in a liquid scintillation counter. Drug doses (shown in FIGS.1A and 1B) were added two hours prior to ConA and IL-2 activation.Background counts were less than 200 cpm. Both CsA and the compoundstested inhibit thymocyte proliferation and activation.

EXAMPLE 24

This example illustrates therapeutic potential for various autoimmuneand inflammatory diseases of the compounds by comparing potency(inhibitive activity) with cytotoxicity data obtained using thefollowing indicative assay procedures. In a mixed lymphocyte reactionassay of compounds nos. 5, 13, 26 and 27, a two-way mixed lymphocytereaction shows a proliferative PMBC response to allogeneic stimulation.Compounds nos. 5 and 26 exhibit the least assay activity in thisspecific, immune-modulating activity assay, having IC₅₀ values exceeding250 μM, as shown in FIGS. 2A and 2B. Both compounds nos. 27 and 13exhibit dose-response activity in this assay, having IC₅₀ 's of 40 and55 μM, respectively, illustrated in FIG. 2C.

FIG. 2D shows a bar graph of IC₅₀ values for five compounds (see abovefor corresponding chemical names) in a mixed lymphocyte assay measuringimmune suppression activity of the compounds. Compound no. 27 did notexhibit significant suppressive activity. Compound no. 28 proved themost potent compound of those assayed.

Cell percent viability in mixed lymphocyte reaction assay culture wasdetermined after six days of cell culture. FIG. 2E shows percentviability bar graph results. Control cells unexposed to drugs aregenerally 78 to 85% viable under these culture conditions. In thisassay, all compounds were present at 100 μM concentrations, generallywell above corresponding IC₅₀ values in this assay (see FIG. 2D). Themost potent compound, compound no. 28, was also the most cytotoxic at100 μM, but this concentration is well above its IC₅₀ value, suggestinga significant therapeutic window. Compounds nos. 13 and 27 exhibitedlittle or no cytotoxicity at concentrations well above their respectiveIC₅₀ values.

Ten additional representative compounds were assayed, showing impressivebiological activity results, in a procedure according to that used inExample 21. IC₅₀ values for tested compounds were obtained in thethymocyte ConA/IL-2, co-stimulation assay, as described above. Fiftypercent (50%) lethal dose concentrations (LD₅₀) for these ten compoundswere obtained using the following cytotoxicity assay.

Human bone marrow-derived stromal cells (early passage) were plated at10⁴ cells/well and allowed to reach confluency following 3 days ofrefeeding. The stromal cells were treated with compounds for 2 hoursprior to washing, and using a viability dye, BCECF, analyzingfluorescence. The highest concentration used was 50 μM (hence, an LD₅₀value greater than 50 μM would indicate no effect at 50 μM). FIG. 2Fillustrates results obtained from both the thymocyte co-stimulationassay (IC₅₀ values) and the cytotoxicity assay (ID₅₀) for compoundsnos.: 10, 35, 39, 42, 43, 45, 49, 50, 51 and 60. As shown, most of thecompounds are non-cytotoxic to stromal cells, yet are very potentproliferation inhibitors in the thymocyte IL-2 co-stimulation assay.

EXAMPLE 25

This example illustrates the effects of several compounds exhibitinginhibitive effects on murine thymocyte proliferation. The data presentedand discussed suggests that the compounds function by mechanismspreviously unknown. In a first part of this example, compounds nos. 30,33, 28 and 27 (see above for chemical names and structures) showeffective inhibition of murine thymocyte proliferation stimulated by ConA and interleukin 1 alpha (IL-1α). Compounds nos. 30, 33, 28 and 27 wereadded to cell cultures two hours prior to activation with Con A andIL-1α in a thymocyte co-stimulation procedure akin to that discussed inexample 21. All compounds tested in the assay exhibited dose-responseinhibitive properties and dose-response curves for each compound wereobtained. IC₅₀ values determined for each compound tested are as follow:compound no. 30 has an IC₅₀ of 0.94 μM, compound no. 33 has an IC₅₀ of8.6 μM, compound no. 28 has an IC₅₀ of 4.6 μM, and compound no. 27 hasan IC₅₀ of less than 12.5 μM. Background counts in the assay were lessthan 200 cpm.

In supplemental investigations, results obtained illustrate that thecompounds utilize different immunosuppressive mechanisms than knownmechanisms of two widely-studied immunosuppressants, CsA and or FK506.In a proliferation assay, mouse thymocytes were pre-incubated overnightwith Con A (a "priming step"), washed, and re-stimulated with IL-2 inthe absence of compounds. On day 4, the-cells were pulsed with tritiatedthymidine and allowed to incubate for an additional 4 hours. The cellswere harvested and the amount of tritiated thymidine incorporated by theharvested cells was determined in a liquid scintillation counter. FIG.3A reports results obtained. In control cells, pre-incubation with Con A"primes" thymocytes by stimulating the CD3 receptor in a manner similarto antigen recognition. Research has shown that CD3 antibody can besubstituted for Con A. When IL-2 was subsequently added, the thymoctyecells proliferated. CsA added during Con A incubation, "priming,"inhibited thymocyte proliferation in response to IL-2 stimulation.However, when thymocytes were pre-incubated with ConA and compound no.35, "priming" occurred, shown by subsequently-observed, normal thymocyteproliferation in response to IL-2 stimulation, as shown in FIG. 3. Thecompounds do not inhibit proliferation by interfering with this "primingstep," necessary for subsequent proliferation in response to IL-2stimulation.

Additionally, the cells were pre-incubated with Con A overnight, washedand stimulated with IL-2 (with or without addition of CsA or compound).On day 4, the cells were pulsed with tritiated thymidine and allowed toincubate for an additional 4 hours. The cells were harvested and theamount of incorporated tritiated thymidine was determined in a liquidscintillation counter. Cells pre-treated with Con A proliferated inresponse to IL-2 addition. FIG. 3B shows results obtained in theseassays. CsA (50 μM) exhibited very little inhibition of thymocyteproliferation (indicated by the amount of incorporated dye recorded). Insharp contrast however, compound no. 35 (at far less concentration, 1μM) dramatically inhibited thymocyte proliferation.

These experiments conclusively indicate that CsA and FK506 (inhibitingCD3 in like manner) have a different action mechanism, as compared withthe compounds. CsA inhibits ConA "priming." The compounds do not. Thecompounds inhibit IL-2 stimulation, CsA does not. The results shownindicate that the compounds would be useful for reducing or preventingside effects from conditions requiring cellular stimulants.

EXAMPLE 26

This example illustrates inhibitive effects of compounds nos. 27 and 28on murine splenocyte proliferation stimulated by anti-mu (10 mg/ml) andinterleukin-4 (IL-4, 12.5 ng/ml). This in vitro assay, described above,is indicative of immune-suppressive/autoimmune treatment assayemphasizing humoral or B cell immune response. The compounds were addedto the cells at the doses indicated two hours prior to activation withanti-mu and IL-4. Both compounds nos. 27 and 28 inhibited splenocyteproliferation in a dose-response manner. IC₅₀ values for compounds nos.27 and 28 were 3.3 μM and 5.2 μM, respectively, as shown in FIG. 4.Background counts were less than 200 cpm.

EXAMPLE 27

This investigation illustrates IL-2 (alpha chain CD25) receptorexpression on mouse splenoyctes. The IL-2 receptor is not expressed onresting T-cells, but is rapidly-induced by certain stimulants, e.g.antigen recognition or treatment with ConA or antibodies to CD3(anti-CD3). IL-2-dependent proliferation requires IL-2 receptorexpression. Splenocytes were stimulated with anti-CD3 antibody (10μg/ml) with or without the addition of CsA (20 μM) or compound no. 49 (1μM). Following overnight incubation, the splenocytes were stained with afluoresceinated anti-IL-2 receptor antibody and fluorescence measured byflow cytometry. FIGS. 5A and 5B are frequency histograms of measurementsfor 20,000 cells per sample. The media control has a low level offluorescence, while stimulation with anti-CD3 stimulates large relativeincreases in IL-2 receptor expression (peak labeled anti-CD3 in FIGS. 5Aand 5B). Co-incubation with CsA inhibits CD3-stimulated, IL-2 receptorexpression, while incubation with compound no. 49, at a concentrationthat blocks 90% of splenocyte IL-2-stimulated proliferation, has noeffect on receptor expression. These data confirm that CsA and compoundno. 49 affect immune cells by different mechanisms.

EXAMPLE 28

This example shows that compounds nos. 49 and 50 did not inhibitproduction of IL-2 in murine thymocytes, in contrast to the effects ofCsA. Thymocytes were stimulated with ConA and IL-1β for 4 days with orwithout adding compounds to the culture media. The supernatants wereremoved and assayed by ELISA for IL-2 levels by a commercially availablekit. The results, shown in FIG. 6, illustrate that CsA incubation at 20nM inhibited IL-2 production and secretion. Also shown, these exemplarycompounds tested did not inhibit IL-2. These data illustrate that CsAand the compounds tested interfere with immune cell function viadifferent mechanisms.

EXAMPLE 29

This example illustrates activity of representative compounds nos. 27,49, 50, 45, 43 or 41 (above) in assays detecting activity in response tocell stimulus. One assay, murine lymph node cell proliferation(stimulated by antigen), was used to determine inhibitive activity ofcompound no. 27 on proliferation of the lymph node cells. This in vitroassay is an immune suppressive/autoimmune treatment predictive assayemphasizing cellular or T-cell immune response. The assay used murineT-cells that proliferate in vitro in response to a soluble proteinantigen, used to prime the T-cells in vivo. Compound was added to thecells at doses indicated in FIGS. 7 and 8 (showing assay results) twohours prior to activation with alloantigen. Compound no. 27 inhibitedT-cell proliferation in a dose-response manner. IC₅₀ values for compoundno. 27 were 23.1 μM for a first experiment (results shown in FIG. 7) and19 μM for a second experiment (results shown in FIG. 8).

Another assay in this example was used to determine inhibitive activityof the compounds on direct IL-2-induced proliferation in a murinecytotoxic T-cell line, CT6. The CT6 cell line is an IL-2-dependent cellline. IL-2 was removed from the medium for 24 hours prior tostimulation. One hour prior to IL-2 stimulation, either CsA or compoundsnos. 49, 50, 45, 43 or 41 were added at various concentrations. Thecells were stimulated with IL-2 and amount of tritiated thymidine dyeincorporated was measured 48 hours later. Background counts were lessthan 5000 cpm. Assay results are graphically represented in FIGS. 9A and9B. The two graphs show that CsA and the compounds have divergenteffects on IL-2-induced proliferation. CsA did not inhibitproliferation, even at very high concentrations. However, in distinctcontrast, the compounds inhibited direct IL-2-induced CT6 proliferation.

EXAMPLE 30

This example illustrates the effects of a reference compound "X", andcompounds nos. 29, 30, 31, 32 and 33 (see above for names andstructures) on yeast growth (Saccharomyces cervisiae)--using 100 μMconcentrations--as compared with yeast activity in the absence of anycompound additive (control). This assay measured anti-yeast andanti-fungal activity of the the compounds tested. As shown in FIG. 10,compounds no. 30 and 33 showed yeast-growth inhibitive activity,predicting that the compounds tested are topical and systemicanti-microbial compounds.

EXAMPLE 31

This example illustrates potential antigen specific anergy-induction ofcompounds. Anergy is a prolonged state of T-cell "unresponsiveness" dueto T-cell antigen recognition (without co-stimulation) or inducedproliferation blockage. This later T-cell anergy may occur when aT-cell's proliferation ability in response to IL-2 is blocked by someagent. Anergy is generally considered to be a type of tolerance toantigen activation. Thus, in vitro anergy is a means for predicting invivo tolerance-enhancing agents. Tolerance is important in preventingorgan rejection in transplant procedures, as well as other autoimmunediseases such as scleroderma, rheumatoid arthritis, lupus, anddiabetes-related autoimmunity.

A B-cell tumor, 2PK3 (H-2d) was used as a stimulating cell for C57BL/6splenocytes (H-2b), a responding cell. Mixed cultures of B-cell tumorand splenocytes were incubated for 5 days with and without 1 μM ofcompound no. 45. After 5 days, the cells were washed and resuspendedwith either media, the original antigen (2PK3) or anti-CD3. Tritiatedthymidine was added to the resulting suspensions and thymidineincorporation measured 24 hours later. Results are shown in FIG. 11. Asillustrated, culture treated with compound no. 45 and untreated culturesresponded equivilantly to anti-CD3. However, cultures incubated withcompound no. 45 for 5 days exhibited a decreased response to primaryantigen, 2PK3. Thus, C57BL/6 splenocytes were inhibited from respondingto an original antigen by the compound used in the pre-incubation step.However, a normal culture response to anti-CD3 stimulation predicts thecompounds exhibited antigen-specific anergy induction properties.

EXAMPLE 32

This example illustrates inhibitive effects of the compounds on humanstromal and Balb/3T3 cell proliferation in response to PDGF stimulation.

This assay is useful for predicting therapeutic activity for preventingor treating restenosis, atherosclerosis and coronary artery disease.Human stromal cells were starved in serum-free media for one day andthen stimulated with 50 ng/ml PDGF-BB. Compounds, at variousconcentrations, were added one hour prior to PDGF stimulation. PDGF andtritiated thymidine were added and the cells allowed to incubate for oneday, following addition of the PDGF and thymidine. 24 Hours later, thecells were harvested and counted by liquid scintillation counting.Results for compounds nos. 28, 30, 31, 33 and 29 are shown in FIG. 12.Background counts (i.e., starved cells) were approximately 1% of controllevels. The results illustrate that the drugs were active in thispredictive in vitro model with IC₅₀ values (in μM) of 0.9, 3.2, 40, >50and >50 for compounds nos. 30, 28, 29, 32 and 31, respectively.

In conjunction with the human stromal cell/PDGF stimulation assay,cytotoxicity of the compounds tested to stromal cells was alsodetermined. FIG. 13 shows results for this cytotoxicity assay. Onlycompound no. 30, exhibiting the most pronounced inhibitive activity ofthe compounds tested, exhibited cytotoxic effects at concentrationsabove 0.9 μM, its IC₅₀ value.

In an assay measuring inhibitive effects in a PDGF/IL-1β co-stimulation,proliferation assay, a group of compounds showed inhibitive properties.The PDGF/IL-1β assay is useful in measuring in vitro activity,indicative of therapeutic potential for treating or preventingrestenosis and reperfusion. FIG. 14 reports IC₅₀ bar graph results for agroup of compounds nos. 27, 28, 29, 30, 31, 32 and 34. In thispredictive, in vitro model, compounds nos. 28 and 30 exhibited potentinhibitive activity, predicting therapeutic applications for restenosisand reperfusion.

The compounds possess inhibitory effects on PDGF-induced proliferationof Balb/3T3 cells. Balb/3T3 cells respond vigorously to PDGFstimulation, and are useful in vitro models for further study ofPDGF-induced proliferation. Disregulated PDGF-proliferative response hasbeen linked to a variety of diseases, including, e.g., restenosis,atherosclerosis, fibrosis, and tumor cell angiogenesis. Cells wereplated in low serum-containing medium for 24 hours prior to stimulationwith various concentrations of compound no. 45. PDGF-BB was added atconstant 10 μM concentrations. Tritiated thymidine was added and cellsharvested for scintillation counting 24 hours later. FIG. 15 illustratesa dose response curve from this assay, including an IC₅₀ value ofapproximately 0.08 μM, exemplifying inhibitory activity of the testedcompound.

EXAMPLE 33

In assays described above and similar to those used in Example 32 andother examples, the ability of various compounds to suppress murinethymocyte and Balb/3T3 cell proliferation in response to selectedstimuli was investigated. In a murine thymocyte ConA/IL-2 co-stimulationassay according to Example 23, IC₅₀ values were obtained for inventivecompound nos. 13, 27, 28, 29, 30, 31, 33, 5 and 26. FIG. 16 reportscomparative results for the different compounds tested in this in vitromodel. Compounds nos. 28, 30, and 27 exhibited the most potentimmune-suppressive effects in these in vitro models.

In another investigation similar to the Balb/3T3 proliferation assay ofExample 32, IC₅₀ and ID₅₀ values for compounds nos. 70, 45, 59 and 58were determined. LD₅₀ values were determined using a cytotoxicity assay,as in Example 32. In these assays, Balb/3T3 cells, stimulated with PDGFand treated with one of the above compounds in a manner identical to thetritiated thymidine procedure above, were incubated instead with aviable dye BCECF, a fluorescent dye. Fluorescence was measured using afluorescent plate reader. The highest concentration used was 50 μM,therefore an LD₅₀ value greater than 50 μM indicates no effect at 50 μM.FIG. 17 illustrates assay results by comparing LC₅₀ value against LD₅₀values for the compounds tested. Most compounds tested werenon-cytotoxic yet were significant inhibitors of proliferation.

EXAMPLE 34

This example compares cytotoxicity results for compound no. 27 fortransformed cells and non-transformed cells. In transformed cells (Ras3T3) and in normal 3T3 cells, cytotoxicity of compound no. 27 atconcentrations of 1, 10 and 100 μM was determined. FIG. 18 reportsresults obtained in this assay. At each of the above concentrations,compound no. 27 was more cytotoxic for the cancer cells than normalcells. These results indicate the compound tested has differentialtoxicity for tumor cells, predicting potential utility inchemotherapeutic cancer treatment.

EXAMPLE 35

This example demonstrates an inhibitory effect on proliferation ofcompound no. 58. One assay was used for investigating effects onPDGF-induced proliferation of human aortic smooth muscle cells (aorticSMC). Cells, purchased from a commercial supplier (Cell Systems, Inc.,Seattle, Wash.), were cultured with various concentrations of PDGF-BBwith and without addition of compound no. 58 (5 μM). As illustrated inFIG. 19, compound no. 58 inhibited PDGF-induced proliferation even athigh PDGF concentrations providing maximum proliferative stimulation. Inaddition, some cultures were treated 1 hour prior to PDGF stimulationwith compound no. 58. As shown, PDGF stimulated proliferation in thiscell line. Addition of 5 μM of compound no. 58 blocked PDGF-stimulatedproliferation and no toxic effects of compound no. 58 were observed.

Another assay was used for investigating inhibitory effects of compoundno. 58 on either basic or acidic FGF-induced proliferation in humanaortic smooth muscle cells (aortic SMC). Disregulated bFGF- oraFGF-induced proliferation is linked to SMC proliferation and neointimalocclusion in atherogenesis and restenosis and plays a role in autocrineand paracrine stimulation of tumor cells and tumor cell-inducedangiogenesis. In this assay, cells were grown in reduced serum (0.5%fetal calf serum) for 24 hours prior to stimulating with variousconcentrations of PDGF. Cells were stimulated with variousconcentrations overnight of either aFGF or bFGF, adding 5 μM of compoundno. 58 in selected cultures. Compound no. 58 was a potent inhibitor ofboth aFGF- and bFGF-induced proliferation in this cell type,representative of other cell types examined. Results shown in FIG. 20A(aFGF) and FIG. 20B (bFGF) illustrate the degree of inhibition. No toxiceffects of compound no. 58 were observed for this cell type in thisassay.

EXAMPLE 36

This example illustrates an investigation of proliferation of murinethymocytes co-stimulated with ConA and IL-2 using a procedure akin tothe procedure in Example 23. In another related assay, inhibitoryeffects on CT6 cell proliferation is examined. CT6 cells are a murineIL-2 dependent, cytotoxic T cell line that proliferate in response tomurine IL-2 (15 U/ml). FIGS. 21A, 21B, 21C and 21D illustrateexperimental results of both compound no. 35 and CsA in each assay. FIG.21A illustrates the extent of inhibitory activity of compound no. 35 onmurine thymocyte co-stimulation and FIG. 21B shows comparativeinhibitory activity of CsA. Both compound no. 35 and CsA exhibitsignificant inhibition of thymocyte proliferation with IC₅₀ values inthe low micromolar and nanomolar ranges, respectively. FIGS. 21C and 21Dillustrate inhibitory effects of compound no. 35 and CsA, respectively.Both compound no. 35 and CsA exhibited no activity in this assay,indicating neither inhibits IL-2-induced proliferation of cytotoxic CT6cells.

EXAMPLE 37

This example illustrates an investigation of inhibitory effects ofcompound no. 58 on Vascular Endothelial Growth Factor (VEGF)-inducedproliferation in a human umbilical vein endothelial cell line (HUVEC).In this assay procedure, cells were grown in reduced serum (0.5% fetalcalf serum) for 24 hours prior to stimulating with variousconcentrations of VEGF. VEGF has been shown to be important in tumorcell-mediated angiogenesis. Compound no. 58, at 5 μM, inhibitedVEGF-induced proliferation at all concentrations of VEGF tested, asshown in FIG. 22.

EXAMPLE 38

This example illustrates an investigation of inhibition of vascular celladhesion molecule (VCAM) expression on HUVEC by compound no. 58. VCAMexpression by endothelial cells is an early event in the pathogenesis ofatherogenesis and multiple sclerosis, among other various autoimmunediseases. FIG. 23 is a series of frequency histograms obtained in thisexemplary assay. The top panel shows a frequency histogram obtained fromflow cytometric analysis of HUVEC cells stained with an antibodydirected against VCAM and a second stem goat anti-mouse-FITC antibody.In the absence of TNFα, VCAM expression on HUVEC was at a very lowlevel. The middle panel shows a frequency histogram of cells stimulatedwith TNFα for 6 hours prior to analyzing by flow cytometry. The averageincrease in cell fluorescence was approximately 10-fold. The bottompanel is a frequency histogram of TNFα -stimulated cells in the presenceof compound no 58. Presence of compound reduced mean fluorescence by afactor of 8, compared with mean fluorescence from TNFα -stimulated cellsin the absence of compound no. 58.

EXAMPLE 39

This example illustrates inhibitive activity of compounds nos. 50 and 57on THP-1 cell adhesion to IL-1β-activated HUVEC. In an investigativeassay, HUVEC were stimulated with IL-1β (10 ng/ml), both in the absenceand presence of varying concentrations of drugs for 8 hours in a 96-wellmicrotiter plate. In the wellplate, human monocytic leukemia cell lineTHP-1 cells were added at 50,000 cells per well. The THP-1 cells werepre-incubated with BCECF, a fluorescence dye that can be used to measurecell number using a fluorescence plate reader. After 10 minutes at 37°C., the microtiter plate was inverted and spun at 900 rpm. The remainingadhering THP-1 cells were then analyzed. As shown in FIG. 24,non-stimulated background adherence was approximately 1500 relativeunits, increasing to approximately 6500 under TNFα stimulation. Thecompounds tested significantly inhibited THP-1 adhesion, even at lowconcentrations.

EXAMPLE 40

This example shows the inhibitory effect of compound no. 58 on eitheraFGF or bFGF-induced proliferation in human pulmonary smooth musclecells. Cells were grown in reduced serum (0.5% fetal calf serum) for 24hours prior to stimulating with various concentrations of PDGF. Cells,stimulated with either aFGF or bFGF, were analyzed in the presence andabsence of compound no. 58 (5 μM). Assay results are reported in FIGS.25A and 25B (aFGF and bFGF, respectively). As shown in the results,compound no. 58 was a potent inhibitor of both aFGF and bFGF-inducedproliferation in this cell type, this pulmonary smooth muscle cell beingrepresentative of other cell types examined. No toxic effect of compoundno. 58 was observed in this assay.

EXAMPLE 41

This example illustrates an ability of compound no. 58 to inhibit VCAM-1or ICAM-1 expression in HUVEC stimulated by TNFα. FIGS. 26A and 26Breport data obtained for compound no. 58 in this assay. These data showthat at concentrations as little as 0.1 μM of compound no. 58, ICAM-1expression is inhibited by more than 80% (i.e., ICAM-1 expression is 20%of a control value). Procedurally this assay is nearly identical to theassay procedure used in Example 38. In this assay, cells were stimulatedfor 8 hours in the presence or absence of compounds and stained withfluorescent antibodies to VCAM-1 or ICAM-1. The resulting ICAM-1 orVCAM-1 expression was analyzed by flow cytometry.

EXAMPLE 42

FIG. 27 illustrates that compound no. 58 inhibited TNFα release using ahuman whole blood ex vivo assay. Using a conventional screeningprocedure, human whole blood was stimulated with mouse TNFα and humanIL-1α with or without various concentrations of compound no. 58. After 6hours, the plasma was analyzed for human TNFα levels using a commercialELISA. Results are reported in FIG. 27.

EXAMPLE 43

This example illustrates conclusive data, compiled from various in vitroand in vivo assay results that the compounds, as represented by aspecies of the disclosed genus (compound no. 50) are anti-cancertherapeutics.

Generally, growth and spread of malignant tumors (rapid cell growth,uncontrolled by normal regulatory mechanisms) characterize cancerdiseases. Precise causes of cancer remain unknown. Preclinical andclinical trials with the compounds indicate that cancer cell growth maybe regulated through the second messenger pathway. Oncogenic mutationsresult in abnormal continuous stimulation of the pathway, leading tounregulated and undifferentiated growth (i.e., malignanttransformation). Cancer cells metastasize (i.e., break through bloodvessels and travel to distant body sites) and secrete enzymes calledmetalloproteases, which "break down" blood vessel walls, allowing thecancer cells to enter the bloodstream and form remote tumors(proteolysis). One such metalloprotease is Type IV collagenase. Inaddition, tumor cell adhesion receptors (integrins) effectattachment--apparently necessary for tumor residence in organs--of tumorcells to blood vessel walls and normal organs. Cancer cells also secretecertain proteins, such as bFGF, that stimulate new blood vesseldevelopment (angiogenesis or neovascularization), these new bloodvessels supplying nutrients fostering malignant tumor growth. Resentresearch results suggest that the second messenger pathway appearsintegral to Type IV collogenase production, adhesion receptorsexpression and bFGF secretion.

Unlike conventional anti-cancer therapies, the compounds, by inhibitingthe second messenger pathway, decrease: tumor cell growth by blockingoncogene-induced events; metastatic potential by blockingmetalloprotease production; tumor adhesion to normal organs by blockingadhesion receptor expression; and a tumor's ability to inducenutrient-carrying blood vessel formation by blocking bFGF or othertumor-dependent growth factor signaling.

The compounds exhibit anti-cancer properties against several malignantconditions, including lung, breast and colon cancer, and unlikeconventional cancer chemotherapy, in vitro, the compounds are non-toxicto normal cells at concentrations lethal to cancer cells. In vitro dataobtained for representative compound no. 50 include the following:

Results obtained for compound no. 50 in the PDGF-induced proliferationof Balb/3T3 cells (Example 32), reported in FIG. 28, illustrate theinhibitive properties of this compound on proliferation induced by PDGF,representing significant inhibition at concentrations as low as 2 μM.This ability to block proliferation (representative of anoncogene-induced event in tumor-cell growth) suggests that thecompounds, specifically compound no. 50, are capable of reducing tumorcell growth by blocking oncogene-induced events.

Another assay measures an ability of the compounds, as represented bycompound no. 50, to inhibit metalloproteinase production in cancercells. In the assay protocol, THP-1 human leukemia cells (1-2×10⁶ /35 mmdish) were plated in RPMI medium with 0.5% serum. Compound no. 50 wasadded at 2.5 μM. Following incubation for 1 hour, TNFα was added andincubated for 18 hours. The supernatants from control and treated plateswere collected and protease activity was determined in gelatin gels(zymogram) after electrophoretic protein separation. As shown in FIG.29, TNFα substantially increased expression of the 92 kD matrixmetalloprotease (MMP) and moderately increased production of the 72 kDMMP. The presence of compound no. 50 blocked the TNFα -stimulatedexpression of both the 92 and 72 kD MMPs. Compound no. 50,representative of compounds of the invention, is capable ofsubstantially reducing metastatic potential of cancer cells by blockingmetalloprotease production.

Other in vitro and in vivo evidence of this potent activity of thecompounds is represented in the following experimental results.

FIGS. 30 shows anti-proliferative activity and FIG. 31 reportsanti-clonogenicity activity of compound no. 50 with HT-29 cells. HT-29cells (1×10⁵ cells/35 mm dish) were plated in McCoy's medium with 10%serum and incubated overnight. Concentrations of 3 and 6 μM of compoundno. 50 were added and viable cell counts made at the times shown. Forclonogenic assays, treated and control cells (300/plate) were plated andallowed to grow colonies. After 7 days the colonies were fixed andcounted. The values in FIG. 31 are the means of 3 plates.

FIGS. 32 and 33 illustrate cytotoxicity and concentration dependence ofcompound no. 50 against 3LL cells. 3LL cells (3×10³ cells/well) wereplated and incubated overnight in RPMI medium containing 10% serum.Compound no. 50 was added at different concentrations and cell numberdetermined at various time points by a vital dye uptake method. Thevalues shown are triplicate of wells.

FIG. 34 shows that compound no. 50, even at much higher concentrationsthan shown having cytotoxicity to tumor cells, lacks cytotoxic activityin normal human bone marrow stromal cells. Human bone marrow stromalcells (1×10⁴ cells/well) were plated in 96 well plates in McCoy's mediumwith serum and incubated overnight. Different dilutions of compound no.50 were added and viable cell counts made by vital dye uptake.

FIGS. 35 and 36 show the effects of compound no. 50 on matrigel invasionand viability in 3LL cells. 3LL cells (4.5×10⁵ cells/well) were platedinto the inner membrane of matrigel chambers. Different concentrationsof compound no. 50 were added to the chamber and incubated for 48 hoursat 37° C. The cells on top of the membrane were removed and cells thatmigrated to the bottom were stained with Diff Quick Solutions and scoredfor relative invasion. The effect of compound no. 50 on viability of 3LLcells at different concentrations was determined separately.

FIG. 37 illustrates VEGF induced proliferation of HUVEC as a predictiveadhesion assay. HUVEC were plated in EBM medium with serum and allowedto grow for 4 days. Different dilutions of compound no. 50 were added tothe plates, followed by VEGF (50 ng/ml) along with tritiated thymidine(1 mCi/mi). Proliferation was measured in quadruplicate and these datashow the effect of compound no. 50 to inhibit adhesion.

FIG. 38 shows the effect of compound no. 50 on THP-1 adherence toIL-1β-stimulated HUVEC. HUVEC (4×10³ cells/well) were plated in RPMImedium with 10% serum and incubated for 48 hours. Differentconcentrations of compound no. 50 were added and incubated for 1 hour.IL-1β (15 ng/ml) was added and incubated for 6 hours. Exponential growthTHP-1 tumor cells, prestained with dye BCECF, were added (1.5×10⁵cells/well) and allowed to adhere for 20 minutes. The number of adheringtumor cells was determined after washing to remove non-adherent cells.

FIG. 39 illustrates the effect of compound no. 50 on VCAM-1 surfaceexpression of TNFα -stimulated HUVEC. This assay is a predictive modelof adhesion. HUVECs grew to 90% confluence in 6 well plates in RPMImedium with 10% serum. Compound no. 50 was added at differentconcentrations and incubated for 30 minutes. TNFα (20 ng/mi) was addedand the cells were incubated for 5 hours. The cells were collected andthe amount of VCAM-1 determined by indirect immunostaining followed byfluorescence activated cell sorter (FACS) analysis. Mean fluorescentintensity of TNFα-stimulated cells was normalized to 100% withdrug-treated samples expressed as a percent of control.

FIG. 40 illustrates the effect of compound no. 50 on ICAM-1 surfaceexpression of TNFα-stimulated HUVEC. This assay is a predictive model ofadhesion. The procedures followed were the same as used in theimmediately preceding assay, except that the cells were stained with anICAM-1 antibody.

FIGS. 41A, 41B and 41C illustrates the results from an in vivo study inmice where B16 melanoma cells were injected intravenously through a tailvein on day 0 and compound no. 50 was administered inter parenterally at10 mg/kg QD or 20 mg/kg QOC, starting days 1-14. The mice weresacrificed on day 15 and the lungs were dissected and fixed in formalin.The number of black metastatic foci were scored and are illustrated inthe three lungs shown in FIGS. 41A, 41B and 41C. In addition bone marrowtoxicity was evaluated by measuring neutrophil and platelet counts inthe mice on day 15. These data are shown graphically below the lungphotographs in each respective figure. These data show that at eitherdose administered compound no. 50 was not toxic to the bone marrow (incontrast to every known cancer chemotherapy regimen), and in fact,increased counts over non-treated control animals.

FIGS. 42A and 42B illustrate T and B cell response assays of micetreated with compound no. 50 in the immediately preceding assayprotocol. Spleens from these treated mice were made into single cellsuspensions in RPMI medium, supplemented with 10% serum, and placed(200,000 cells/well) in flat-bottomed 96 well plates. Anti-CD3 (FIG.42A) or a mixture of an anti-mu/IL-4 (FIG. 42B) were added to the wellsat final concentrations of 1 mg/ml and 1 mg/ml/12.5 ng/ml, respectively.Appropriate positive and negative controls were set up on each plate andall samples were assayed in quadruplicate. The plates were incubated for2 days and proliferation was measured by tritiated thymidineincorporation.

FIG. 43 reports results in an in vivo experiment showing that compoundno. 50 can arrest growth of Lewis Lung Carcinoma in mice. BDFI mice wereinjected subcutaneously with 1×10⁶ 3 LL cells on day 0 and then treatedwith compound no. (20 mg/kg i.p.), cyclophosphamide (20 mg/kg) orvehicle on alternate days beginning on day 7. The animals weresacrificed on day 20 and the lung tumors dissected and weighed. FIG. 43illustrates that compound no. 50 showed superior results over anexisting cancer chemotherapeutic agent.

FIG. 44A and 44B illustrates platelet and neutrophil counts,respectively, of the sacrificed mice in the immediately preceding assay.In addition, platelet and neutrophil counts in the mice were not alteredfrom vehicle, indicating that bone marrow was not a target of toxicityfor compound no. 50.

FIGS. 45A, 45B and 45C illustrate a photographic comparison of lungsfrom the 3LL exposed mice with or without treatment using compound no.50. The lungs were injected with india ink such that normal lung tissuestains black and tumors appear white. The difference in lungs treatedseven days after tumor cell administration was visually apparent. Thesein vivo data are highly predictive of significant clinical activity andreduced bone marrow toxicity of the compounds.

EXAMPLE 44

This example illustrates conclusive, in vitro and in vivo evidencepredicting that the compounds, as represented by a species of thedisclosed genus (compound no. 45), are effective therapies for treatingautoimmune diseases and suppressing an immune response.

One such autoimmune disease, rheumatoid arthritis, is triggered byunknown environmental or endogenous events in a genetically susceptibleindividual. Dysregulation of B lymphocytes leads to the production ofimmune complexes. Activated T cells in involved joints (predominantly ofthe CD4 helper type) promote autoantibody production. Macrophages anddendritic cells produce large numbers of inflammatory cytokines, furtherstimulating lymphocytes. In addition, both lymphocytes and macrophagesproduce other cytokines which stimulate proliferation of synovial cellsleading to "pannus" formation and joint deformity. Degradative enzymessuch as type IV collagenase are released into the joint space anddestroy connective tissue including the cartilage on articular surfaces.Lymphocyte- and macrophage-released cytokines play various roles in thepathogenesis of joint destruction and systemic symptoms in rheumatoidarthritis. Compounds that affect only a single component of this complexprocess are unlikely to be effective or disease-modifying unless thesecompounds target a process that is both proximal in the cascade of andfundamental to the inflammatory process itself. Because biologic systemsappear invariably redundant, conventional treatments target a singleaspect of a complex reaction and are thus only partially effective.

The compounds interrupt many key components of the cascade of eventsthat lead to both joint destruction and the systemic complications ofrheumatoid arthritis. Specifically in the following in vitro and in vivoassays, compound no. 45 (as a representative compound of the invention)inhibited: T and B cell proliferation, thus inhibiting abnormalautoantibody production; macrophage activation, cytokine production, andmost importantly, signaling by multiple cytokines (for example, T and Bcell driven proliferation in response to IL-2, IL-4, IL-7, TNFα and Tcell receptor activation by antigen); proliferative signals to synovialcells in response to PDGF, FGF, EGF, and insulin-like growth factors(IGF); adhesion molecule expression (including VCAM and ICAM),stimulated by local inflammation, a suppression of these adhesionmolecules likely leading to a decrease in lymphocyte and macrophagetrafficking to the inflammation site, thus decreasing amplification ofthe inflammatory process. Therefore, the compounds, represented bycompound no. 45, inhibit, from multiple points, the inflammatory anddysregulated immune response resulting in acute and chronicsymptomatology of diseases such as rheumatoid arthritis.

In vitro and in vivo data confirm activity of the compounds as animmunosuppressive therapeutic, as represented in the following results.

CT-6.1 cells were starved overnight and stimulated with IL-2 (20 ng/ml).Twenty hours later, cells were labeled for 4 hours with ³ H-TdR andcounted by liquid scintillation. Background counts were approximately2000 CPM. The IC50 for inhibition in this cell line was approximately0.8 μM. CsA, used as a negative control, had no effect on IL-2-mediatedproliferation. FIG. 46 reports results obtained in this assay,illustrating inhibition of proliferation by compound no. 45, as measuredby ³ H-TdR incorporation using cell counts.

CT6.1 cells were cultured with IL-2 with or without the addition ofcompound no. 45 (2 μM) and viable cells counted for up to 8 days. On day4, two cultures previously incubated with compound no. 45, were washedand recultured with fresh IL-2 with or without compound no. 45 (2 μM).Results reported in FIG. 47 illustrate that compound no. 45 inhibitsproliferation as measured by cell number, its effect being reversibleeven after 4 days in culture.

The results shown in FIGS. 48A, 48B, and 48C illustrate that compoundno. 45 also inhibited mitogenic responses to IL-2, IL-4 and IL-7.Procedurally, CT-6.1 cells were starved overnight and stimulated witheither IL-2 (20 ng/ml), IL-4 (50 ng/ml ) or IL-7 (25 ng/ml). ³ H-TdR wasadded at 20 hours, cells were harvested 4 hours later, and theincorporation of ³ H-TdR was determined. Compound no. 45 illustratedIC50's<1.0 μM for IL-2, IL-4 or IL-7-stimulated proliferation of CT6.1cells. All of the incorporation studies were confirmed with parallelcell counting experiments (data not shown). These data predict that thecompounds induce immunosuppression similar to an X-linked SCID-likecondition.

In another in vitro protocol, thymocyte proliferation was induced bysub-mitogenic doses of Con A (0.25 μg/ml) and IL-2 (20 ng/ml). Cellswere stimulated for 96 hours, prior to addition of ³ H-TdR for 4 hours.The cells were harvested and incorporation of ³ H-TdR was determined.Background counts were approximately 2000 cpm. In the results reportedin FIG. 49, compound no. 45 also inhibited murine thymocyteproliferation in response to ConA and IL-2, with an IC₅₀ ofapproximately 0.35 μM.

FIGS. 50A and 50B illustrate that compound no. 45 inhibits a murinemixed tumor lymophocyte culture (MTLC) and a human mixed leukocytereaction (MLR). In the MTLC, splenocytes were stimulated (co-culture)with an alloantigen, B cell tumor target cell line (2PK3) in thepresence or absence of compound no. 45. Cells were stimulated for 3days, ³ H-TdR was added and the cells were harvested 4 hours later.Incorporation of ³ H-TdR was determined by liquid scintillation.Background counts were 1000-1200 CPM. For the human MLR, purifiedperipheral lymphocytes from two HLA disparate individuals wereco-cultured for 6 days with or without compound no. 45. On the 7th daythe cultures were pulsed with ³ HTdR for 24 hours and counted byscintillation. As reported in FIGS. 52A and 52B, an IC₅₀ for the MTLCand MLR were both approximately 0.5 μM.

FIG. 51 illustrates an effect of the delayed addition of compound no. 45on co-stimulated thymocyte proliferation. In an assay procedure, murinethymocytes were stimulated with ConA and IL-2 with 1 μM compound no. 45added either 1 hour prior to IL-2 (-1), simultaneous with IL-2 (0) or atvarious times following IL-2 addition (1-92 hour). At 92 hours thethymocytes were pulsed with ³ H-TdR, harvested and counted byscintillation 4 hours later. The results reported in FIG. 53 illustratethat compound no. 45 maximally inhibited thymocyte proliferation evenwhen added 24 hours following the IL-2 stimulation. Furthermore,significant inhibition remained even at 72 and 92 hours followingaddition of IL-2.

FIG. 52 illustrates that compound no. 45 inhibited anti-CD3 stimulatedsplenocyte proliferation. In the assay, murine splenocytes wereactivated with a monoclonal antibody directed against CD3 (1 μg/ml),resulting in an IL-2 mediated proliferative response. Background wasapproximately 2000 cpm. As reported in FIG. 52, the IC50 for inhibitionin splenocytes was approximately 0.85 μM.

FIGS. 53A and 53B illustrate that compound no. 45 did not inhibit T cellreceptor (CD3) mediated signaling. Procedurally, murine splenocytes wereincubated overnight with anti-CD3 in the presence or absence of CsA (0.1μM) or compound no. 45 (1 μM)--FIG. 53A. Following overnight incubation,cells were washed and restimulated with IL-2 for 20 hours withoutaddition of CsA or compound. The cells were pulsed with ³ H-TdR for 4hours and harvested and incorporation of ³ H-TdR determined.Preincubation of splenocytes with CsA together with anti-CD3 blocks theability of the cells to respond to IL-2. This is due to the fact thatCsA inhibited TCR mediated up-regulation of the IL-2 alpha chainreceptor (CD25). In contrast, preincubation of the splenocytes with thecompound and anti-CD3 did not block the splenocyte's ability to respondto IL-2.

If the cells were first treated with anti-CD3 overnight, washed andrecultured using IL-2 with CsA (0.1 μM), proliferation was not inhibitedcompared to the IL-2 only control--FIG. 53B. CsA did not block IL-2mediated signaling events. However if the anti-CD3 "primed" splenocyteswere stimulated with IL-2 in the presence of compound no. 45 (1 μM),proliferation was inhibited. These data demonstrate that compound no. 45specifically inhibited IL-2 induced proliferation of anti-CD3 activatedsplenocytes, without blocking TCR-mediated activation, thereforepredicting a distinctly different mechanism of action than CsA.

In another assay, compound no. 45 did not inhibit anti-CD3 mediatedupregulation of the IL-2 receptor alpha subunit. These data arepresented in the histograms in FIGS. 54A and 54B. Murine splenocyteswere activated for 24 hours with anti-CD3 (1μ/ml), stained with afluorescent antibody to CD25 and analyzed by flow cytometry. FIG. 54A isa histogram for cells treated with or without 100 nM CsA or the mediacontrol. CsA inhibits CD-25 receptor upregulation by inhibiting TCRsignaling. However, in FIG. 54B, a similar experiment is shown, exceptthat the cells were treated with compound no. 45 (1 μM) rather than CsA.Results reported confirm that compound no. 45 did not inhibit CD-25(p55) receptor upregulation by anti-CD3.

In FIG. 55, compound no. 45 did not inhibit IL-2 receptor beta (p70)subunit internalization and cell surface down-regulation following IL-2stimulation. In this assay protocol, CT-6.1 cells were starved overnightand stimulated with 20 ng/ml IL-2. At various times following IL-2stimulation, cells were rapidly suspended in ice cold PBS and stainedusing a fluoresceinated monoclonal recognizing the beta subunit of theIL-2 receptor and cell surface expression analyzed by flow cytometry.FIG. 55 is a plot of mean fluorescence versus time (in minutes)following IL-2 addition, illustrating that IL-2r beta subunit wasrapidly internalized following IL-2 addition with cell surfaceexpression remaining low for 6 hours. Compound no. 45 did not inhibitthis receptor internalization activity.

As shown in FIGS. 56A and 56B, compound no. 45 also induces antigenspecific T-cell anergy. Procedurally, murine splenocytes were stimulatedwith an alloantigen target cell line 2PK3 in the presence or absence ofcompound no. 45 (1 μM; approximate IC₅₀). Following 5 days in culture,the cells were washed, re-cultured and re-stimulated with the originalpriming antigen and anti-CD3 monoclonal antibody. The secondary responseto the priming antigen was inhibited if the cells were cultured withcompound no. 45 during the five day primary culture. The polyclonalT-cell response to anti-CD3 was not affected, indicating that thecompound was not cytotoxic during the 5 day priming period. Althoughcells pretreated with compound no. 45 could respond normally topolyclonal stimulation, those that were challenged with the alloantigencould not. Therefore, compound no. 45 induced a specific state ofunresponsiveness or anergy to the alloantigen.

In FIG. 57, results shown illustrate that compound no. 45 inhibits Bcell proliferation. Murine splenocytes were stimulated with anti-IgMantibodies (10 μg/ml) and murine IL-4 (12.5 ng/ml). Cells were pulsedwith ³ HTdR at 44 hours, harvested 4 hours later, and the incorporationof ³ H-TdR was determined by liquid scintillation. As shown in FIG. 59,the compound inhibited proliferation, with an IC₅₀ of 1.2 μM.

FIGS. 58A and 58B illustrate that compound no. 45 did not inhibitCD28-mediated IL-2 release. Murine splenocytes were stimulated with amixture of anti-CD3 and anti-CD28 monoclonal antibodies. Compound no. 45inhibited T-cell proliferation in this system (FIG. 60A) but did notinhibit the CD28-mediated release of IL-2 (FIG. 60B).

FIGS. 58C, 58D, and 58E illustrate that compound no. 45 inhibited IFN-γrelease by blocking IL-2 signaling. Procedurally, murine splenocyteswere stimulated with a mixture of anti-CD3/CD28 antibodies. Compound no.45 inhibited release of IFN-γ (FIG. 60C). Addition of an anti-IL-2receptor alpha subunit antibody and an anti-IL-2 receptor beta subunitantibody to the cultures inhibited both proliferation and the release ofIFN-γ (FIG. 60D). This demonstrates that the inhibition of IFN-γ releaseafter stimulation with anti-CD28/CD3 was due to blocking an IL-2 signal.

FIGS. 59A, 59B and 59C report assay results investigating the effect ofcompound no. 45 on cytokine release from anti-CD3-stimulated mousesplenocytes. Cells were stimulated with anti-CD3 overnight with orwithout either CsA (50 nM) or compound no. 45 (1 μM). Concentrationschosen were approximately equal to IC₅₀ values. The supernatants wereharvested and assayed for cytokine levels using commercial ELISAs forproliferation. Anti-CD3 stimulated release of IL-2, IFN-γ and IL-4(background levels for media control were negligible). As shown in FIG.59A, compound no. 45 did not significantly inhibit IL-2 release, incontrast to CsA. However, compound no. 45 drastically inhibited IFN-γrelease similar to CsA (FIG. 59B), predicting that the compound had adifferential effect on Th-2 cell cytokine release, which secrete bothIL-2 and IFN-γ. Compound no. 45 only partially inhibits IL-4 release,which is produced by Th-2 cells (FIG. 59C).

In an assay related to the MTLC assay, cytokine levels were measured byELISA from the supernatants of an MTLC culture. FIG. 60A showsinhibition of overall proliferation with compound no. 45. FIGS. 60B and60C illustrate that compound no. 45 did not inhibit either IL-2 or TNFαrelease from the MTLC. In contrast, FIG. 60D shows that compound no. 45inhibited IFN-γ release.

FIG. 61 illustrates that compound no. 45 did not have an effect onprostaglandin E2 release from IL-1α-stimulated human foreskinfibroblasts, HS68. Cells were stimulated with 100 pg/ml IL-1α. Compoundno. 45 was added 1 hour prior to stimulation. Supernatants wereharvested 24 hours later and levels of PGE2 were analyzed by commercialimmunoassay. The results are reported in FIG. 61 and confirm no effecton PGE₂ release.

Compound no. 45 inhibited adhesion of monocytic leukemia cells (THP-1)to IL-1β or TNFα-activated HUVEC. HUVEC were stimulated with 20 ng/mlIL-1β or 15 ng/ml TNFα for 6 hours. Fluorescence labeled (BCECF-AM)THP-1 cells were allowed to adhere to the HUVEC for 20 minutes, washedonce and the amount of fluorescence remaining analyzed on a fluorescenceplate reader. The results, reported in FIGS. 62A and 62B (TNFα or IL-1β,respectively), show inhibition of THP-1 adhesion to HUVEC.

Assay results reported in FIGS. 63A and 63B, illustrate that compoundno. 45 inhibited adhesion receptor expression on HUVEC. HUVEC werestimulated with TNFα (20 ng/ml) for 5 hours and stained usingfluorescent antibodies to ICAM-1 or VCAM-1. Cell surface expression wasanalyzed by flow cytometry. All values were normalized to the peak meanfluorescence of the positive control (=100%). Average levels ofinduction using TNFα-stimulation were 20-fold higher for ICAM-1 and over50-fold higher for VCAM-1 from that of nonstimulated controls, as shownin FIGS. 63 and 63B.

FIG. 66 reports that compound no. 45 inhibited PDGF-induced murineBALB/3T3 proliferation. Procedurally, cells were rested overnight in0.2% serum and stimulated with 25 ng/ml PDGF. Compound no. 45 was added1 hour prior to stimulation. Cells were pulsed with ³ H-TdR 24 hourslater and harvested for scintillation counting 4 hours later. As shownin FIG. 64, compound no 45 inhibited proliferation in this assay atconcentrations less than 30 μM.

Collectively, the foregoing data substantially support the conclusionthat compound no. 45, representative of the compounds disclosed herein,is a good immunosuppressive and anti-inflammatory therapeutic, effectingmany of the cascading events characteristic of diseases such asrheumatoid arthritis.

EXAMPLE 45

This example illustrates in vitro evidence predicting that thecompounds, as represented by a specie of the disclosed genus (compoundno. 58), are effective therapies for atherosclerosis and restinosis.

The pathologic mechanisms leading to restenosis mimmic processesobserved in atherosclerosis, yet at an accelerated pace. Arterialnarrowing resulting from atherosclerosis is the end result of a complexprocess involving injury to blood vessels, initiated by subintimalaccumulation of lipids triggering a cascade of cellular and cytokinemediated events. Such events include accumulation of platelets andinflammatory cells at the site of injury. Cytokines are released whichstimulate smooth muscle cell proliferation. The arterial narrowingobserved is predominantly due to the localized accumulation ofmacrophages and proliferation of smooth muscle cells within the arterialwall. Unlike the slow chronic narrowing seen in atherosclerosis, thedisease process is greatly accelerated after arterial injury caused byangioplasty or vascular surgery. In the assay data which follows, thecumulative reported results illustrate that compound no. 58,representative of the compounds, inhibits many of the cellular andcytokine-mediated events that lead to atherosclerosis and restenosis.

FIGS. 65A-65F illustrate inhibition of proliferation in either humanaortic or pulmonary smooth muscle cells (SMC) by compound no. 58.Procedurally, cells were cultured in 0.5% serum containing medium 24hours prior to stimulation with various concentrations of either PDGF(FIGS. 65A and 65B), acidic FGF (FIGS. 65C and 65D) or basic FGF (FIGS.65E and 65F). Cells were stimulated for 24 hours prior to labeling with³ H-TdR for 4 hours and harvested and counted by scintillation. Compoundno. 58 was added 1 hour prior to stimulation at a concentration of 5 μM.Even at points of maximum stimulation of the growth factors, compoundno. 58 completely inhibited cellular proliferation in both aortic andpulmonary SMC. In addition cell number was assessed and those treatedwith compound no. 58 showed no increase in cell number following growthfactor stimulation.

FIGS. 66A and 66B are a dose response and cytoxicity curves,respectively, for inhibition of proliferation in Balb/3T3 cells bycompound no. 58. In the assay protocol, cells were starved overnight in0.5% serum containing medium, followed by stimulation with 20 ng/ml PDGFfor 24 hours. The cells were labeled with ³ H-TdR for 4 hours andharvested and counted by scintillation. The IC₅₀ for inhibition in thiscell line was approximately 200-500 nM, the results being reported inFIG. 66A. FIG. 66B illustrates that compound no. 58 was not cytotoxic toBalb/3T3 cells.

As illustrated in FIGS. 67A and 67B, compound no. 58 inhibitedVEGF-induced proliferation in HUVEC and EGF-induced proliferation inSwiss/3T3 cells, respectively. Procedurally, HUVEC were placed in 0.5%serum containing medium prior to stimulation with various concentrationsof VEGF, with or without compound no. 58 (5 μM). Twenty four hours laterthe cells were pulsed with ³ H-TdR and 4 hours later harvested andcounted by scintillation. The IC₅₀ for inhibition was approximately50-100 nM. Swiss 3T3 cells were stimulated with 20 ng/ml EGF and 24hours later harvested and counted by scintillation. The IC₅₀ forinhibition was approximately 20 nM.

FIG. 68 illustrates an effect of the delayed addition of compound no. 58to Balb/3T3 proliferation in response to PDGF-BB. Cells were stimulatedwith PDGF (20 ng/ml) and compound no. 58 added either simultaneous withPDGF (0) or at various times after addition of PDGF, up to 6 hourslater. At 24 hours, the cells were pulsed with ³ H-TdR and counted byscintillation four hours later. Further experimentation has shown nearlycomplete inhibition in proliferation, even if compound no. 58 was addedas late as 20 hours after PDGF.

FIG. 69 illustrates that compound no. 58 inhibited PDGF-inducedproliferation in Balb/3T3 cells to a greater extent than serum-inducedproliferation. In the assay protocol, cells were serum starved for 24hours before adding either 20 ng/ml PDGF or 10% serum. The cells werepulsed with ³ HTdR 24 hours later. Compound no. 58 did not have asignificant effect on serum-induced Balb/3T3 proliferation.

As shown in FIG. 70, endothelial cell migration is inhibited by compoundno. 58. HUVEC were placed in a Matrigel invasion assay system (BectonDickinson) and VEGF-induced migration assessed with or without varyingconcentrations of compound no. 58. Matrigel migration of HUVEC to 50ng/ml VEGF was inhibited in a dose dependent manner, with an IC₅₀ ofapproximately 100-200 nM.

FIG. 71 illustrates that compound no. 58 did not inhibit chemotaxis ofhuman smooth muscle cells (SMC) to PDGF. In the assay, cells were seededin a Boyden chamber with or without various concentrations of compoundno. 58. Eight hours later, the number of cells that had migrated werescored visually. Compound no. 58 had no effect on PDGF-directed SMCchemotaxis.

FIGS. 72A and 72B illustrate that compound no. 58 inhibited THP-1 celladhesion to either TNFα or IL-1β-stimulated HUVEC, respectively.Procedurally, HUVEC were stimulated with TNFα or IL-1β for 8 hours andTHP-1 cells added for 20 minutes. The HUVEC were then washed andanalyzed for adherence. Compound no. 58 inhibited adhesion of THP-1cells to HUVEC with an IC₅₀ of approximately 1 μM for both TNFα andIL-1β.

EXAMPLE 46

This example illustrates an ability of the compounds to inhibitIL-12-mediated interferon gamma (IFNγ) production. The following assaysare predictive of activity of the compounds nos. 45 and 76 asimmunosuppresive agents. C57BL/6 splenocytes were induced to secreteIFNγ by addition of exogenous IL-12. Compounds nos. 45 and 76 weretitrated into the cultures over a 0.01 to 10 μM range. The amount ofIFNγ in the culture supernatants after 48 hours was quantitated by acommercially available ELISA. Techniques employed for culturepreparation, compound addition, washing, supernatant isolation andconcentration conform with procedures normally employed by skilledartisans in screening compounds for immunosuppressive activity. DMSO wasused as a solvent for delivery of the compounds to cell cultures.Results obtained in the assay are provided in Table III below.

                  TABLE III                                                       ______________________________________                                        Compound/          IFNγ(ng/ml)                                          (Concentration, μM)*                                                                          Exp 1  Exp 2                                               ______________________________________                                        control/50 U IL-12 6.0    19.1                                                Vehicle            --     13.1                                                45/(10)            <0.2   <0.2                                                45/(1.0)           5.5    6.8                                                 45/(0.1)           5.8    15.1                                                45/(0.01)          --     14.9                                                76/(10)            <0.2   <0.2                                                76/(1.0)           --     0.2                                                 76/(0.1)           --     15.0                                                76/(0.01)          5.5    13.7                                                ______________________________________                                         *All cultures contained 50 U IL12                                        

As shown in the above table, the IC₅₀ value for compounds nos. 45 and 76are between 10 μM and 1.0 μM (no. 45) and 1.0 μM and 0.1 μM,respectively. These values are within concentrations which may beattained in vivo.

EXAMPLE 47

This example illustrates an ability of the compounds to inhibit bothIL-1α or IL-6-stimulated proliferation of D10(N4)M or B9 cells,respectively. Using procedures similar to those discussed in theforegoing examples, cultures of D10(N4)M and B9 cells were incubatedwith 10 pg/ml (approximately 5 U/ml) of IL-1α or 10 pg/ml (approximately3 U/ml) of IL-6, respectively. DMSO was used as a solvent for deliveryof the compounds to cell cultures. Compounds nos. 45, 58 and 76 wereadded at concentrations of 10, 1.0, 0.1 and 0.01 to each series ofcultures, set up with appropriate, corresponding controls. Resultsobtained in the assays are provided in Tables IV D10(N4)M! and V B9!below:

                  TABLE IV                                                        ______________________________________                                        D10(N4)M                                                                      Compound/                                                                     (Concentration,                                                                        Inhibition of Proliferation (%)                                      μM)*  Compound no. 45                                                                           Compound no. 58                                                                           Compound no. 76                              ______________________________________                                        10 μM 100         100         100                                          1.0 μM                                                                              100         0           72                                           0.1 μM                                                                               0          0           0                                            0.01 μM                                                                              0          0           0                                            ______________________________________                                    

                  TABLE V                                                         ______________________________________                                        B9                                                                            Compound/                                                                     (Concentration,                                                                        Inhibition of Proliferation (%)                                      μM)*  Compound no. 45                                                                           Compound no. 58                                                                           Compound no. 76                              ______________________________________                                        10 μM 100         100         100                                          1.0 μM                                                                              100         0           100                                          0.1 μM                                                                               73         0            0                                           0.01 μM                                                                              0          0            0                                           ______________________________________                                    

These data indicate that compounds 45, 58 and 76 can block anIL-1α-mediated proliferative response in D10(N4)M with IC₅₀ 's between0.1-1.0 μM for compound no. 45 and 76 and between 1.0-10 μM for compound50. Similarly, compound nos. 45 and 76 inhibit IL-6-mediated growth ofB9 cells with IC₅₀ 's between 0.1-1.0 μM, and for compound no. 58,between 1.0-10 μM. All these concentrations are achievable in vivo.

EXAMPLE 48

This example is illustrates in vivo activity of the compounds in apredictive mouse hemolytic plaque assay (a model of B-cell activation).Compounds nos. 58 and 76 were evaluated for their ability to inhibit invivo antibody formation to SRBC shown using a plaque-forming assay.

Procedurally, CD-1 (ICR) female mice (Charles River), 10-14 weeks of agewere sensitized intraperitoneally with 1.25×10⁸ sheep red blood cells(SRBC) in 0.2 ml saline. The mice were divided into groups of 508.Compound administration commenced on the day of sensitization andcontinued daily through day 3. Control mice received a coequal volume ofvehicle. On the fourth day after sensitization, the mice weresacrificed, each spleen excised, a splenocyte suspension prepared byhomogenization in 4 ml Hank's Buffered salt solution (HBSS), and anucleated cell count (WBC) determined by Coulter counter. A splenicsuspension with SRBC absorbed guinea pig complement was blended in a0.5% agar solution. Two 0.1 ml aliquots were dispersed onto a petri dishand a monolayer formed by dispersion of each aliquot beneath a 22 mmcover slip. The dishes were incubated at 37° C. in 5% CO₂ for 2-2.5hours. Plaques were counted with a dissecting microscope.

Data obtained in the assay are reported below in Table VI. Propyleneglycol is the vehicle for all groups treated, with the exception ofgroup #1, which was left untreated. Group #1 was immunized with SRBClike all other treated groups, but was not treated with propylene glycolor compounds. Group #2, the vehicle test group (in the absence of anycompound) was used to verify any inhibitive or exacerbative plaques.From the data shown, it appears that the propylene glycol vehicle has aninhibitory effect on the subjects in group #2. All compounds were giventwice daily at 7:30a.m. and 5:30p.m. For example, the subjects in group#3 were dosed 25 mg/0.1 ml/mouse in the morning dose and received thesame dose in the evening for a total of 50 mg/ml daily.

                  TABLE VI                                                        ______________________________________                                                                   PFC/        WBC/  Percent                                               Dose  Spl ×                                                                        Percent                                                                              Spl ×                                                                         Chg                              Grp  Treatment                                                                              #/Grp  mg/kg 10.sup.3                                                                           Inhibition                                                                           10.sup.3                                                                            WBC                              ______________________________________                                        1    Untreated                                                                              10     --    372  --     183   --                               2    RS-VEH   5       0.0  321  --     174   --                               3    Comp.    5      50.0  190  41     161   -7                                    no. 58                                                                   5    Comp.    6      12.5  283  12     207   19                                    no. 58                                                                   9    Comp.    7      50.0   29  91     171   -2                                    no. 76                                                                   11   Comp.    3      12.5  221  31     155   -11                                   no. 76                                                                   ______________________________________                                    

As the above data report, compound no. 58 inhibits the immune responseby about 41 percent at a concentration of 50.0 mg/kg and about 12percent at a concentration of 12.5 mg/kg. Compound no. 76 inhibits theimmune response by 91 percent at a concentration of 50.0 mg/kg and 31percent at a concentration of 12.5 mg/kg.

EXAMPLE 49

This example illustrates an ability of the compounds to inhibit 6TIrasbladder carcinoma cell-induced angiogenesis in chorio-allantoic membrane(CAM) of developing chick embryos. Tumor growth is dependent onneovascularization of angiogenesis mediated by several growth factors,including basic fibroblast growth factor (bFGF), vascular endothelialgrowth factor (VEGF), and epidermal growth factor(EGF), which aresecreted by tumors. Inhibitors of angiogenesis, such as fumigalin andsuramin, possess anti-tumor activity in animal tumor models.Angiogenesis induced by tumors in the chorioallantoic membrane (CAM) ofdeveloping chick embryos is one established in vivo model systemeffective in studying the effects of agents that inhibitneovascularization. This example tests the ability of the compounds toinhibit angiogenesis in vivo.

Procedurally, the surface of 6 day-old postertilization chicken eggs(white leghorn, NCSU poultry science) was sterilized with wescodyne, andthe CAM was exposed by cutting a window (1 cm²) on one side of the eggusing a false airsac technique as described in Ausprunk et al.,"Vascularization of Normal Neoplastic Tissues Grafted to ChickenChorioallantois," Am. J. Path., Vol. 79, pp.597-618 (1975). After 24-48hours, the 6TIras, bladder carcinoma cells, without compound (i.e.,positive controls) and with compounds nos. 50 and 58 were placed on theexposed CAM, the windows were sealed with a transparent tape, and theeggs were incubated in a humid incubator at 35° C. Eggs were examined atintervals for a period up to 6 days post-inoculation, using astereoscopic dissection microscope. Positive angiogenesis was scoredbased on the development of more than 5 loops of blood vesselsdelineating the added cells or bFGF. The respective compounds, dissolvedin appropriate solvent, were absorbed on to 1×1×2 mm pieces of gelfoamsponges and placed on the CAM along with tumor cells and observed forsurrounding inhibition zones (avascular areas). The results areexpressed as the number of angiogenes in positive eggs/total number ofeggs and the percentage of eggs showing inhibition of angiogenesis(percentage of positive).

Data obtained in this in vivo assay are presented Table VII below. Themedia control group (DMEM media only) were 0/6 positive forangiogenesis. The control group (6TIras cells alone) were 6/6 positivefor angiogenesis with 0% inhibition. The positive control group(Suramin) were 2/8 positive for angiogenesis at 1 mM with 75%inhibition. In the treatment group using compound no. 50, there appeareda dose-dependent inhibition of angiogenesis. An IC₅₀ of 800 nM yielded4/8 positive for angiogenesis with a corresponding 50% inhibition. Inthe treatment group using compound no. 58, a dose-dependent inhibitionof angiogenesis was also observed. An IC₅₀ of 100 nM resulted in a 3/6positive for angiogenesis with a corresponding 50% inhibition. Thesedata confirm that the compounds tested, representative of compounds ofthe invention inhibit angiogenesis in this predictive in vivo model havetherapeutic potential as growth inhibitors of cancerous tumors.

                  TABLE VII                                                       ______________________________________                                                                # Positive Percent                                    Group  Treatment        Angiogenesis                                                                             Inhibition                                 ______________________________________                                        1      Control (DMEM)   0/6        0                                          2      GTIras (cells)   6/6        0                                          3      Suramin (1 mM)   2/8        75                                         4      Compound no. 50/100 nM                                                                         6/7        14.3                                       6      Compound no. 50/800 nM                                                                         4/8        50                                         7      Compound no. 50/2000 nM                                                                        3/7        67                                         8      Compound no. 58/100 nM                                                                         3/6        50                                         9      Compound no. 58/500 nM                                                                         3/7        57                                         10     Compound no. 58/1000 nM                                                                        2/5        60                                         11     Compound no. 58/5000 nM                                                                        1/6        83.4                                       ______________________________________                                    

What is claimed is:
 1. A method for preparing a compound, includingresolved enantiomers and/or diastereomers, hydrates, salts, solvates andmixtures thereof, the compound having a straight or branched aliphatichydrocarbon structure of formula I: ##STR108## wherein: n is an integerfrom one to four;m is an integer from four to twenty; independently, R₁and R₂ are hydrogen, a straight or branched chain alkyl, alkenyl oralkynyl of up to twenty carbon atoms in length or --(CH₂)_(w) R₅, wbeing an integer from one to twenty and R₅ being an hydroxyl, halo, C₁₋₈alkoxyl group or a substituted or unsubstituted carbocycle orheterocycle; or jointly R₁ and R₂ form a substituted or unsubstituted,saturated or unsaturated heterocycle having from four to eight carbonatoms, N being a hetero atom; R₃ is hydrogen or C₁₋₃ ; or jointly one ofR₁ or R₂ and R₃ form a substituted or unsubstituted linking carbonchain, having from one to four carbon atoms, joining the O and N in acyclic structure, an integer sum equal to n+a number of carbon atoms inthe linking carbon chain being less than six; a total sum of carbonatoms comprising R₁ or R₂, (CH₂)_(n) and (CH₂)_(m) does not exceedforty; and R₄ is a terminal moiety comprising a carbocycle orheterocycle having one ring or two-fused rings, each ring having five orsix ring atoms, wherein a ring atom of the terminal moiety is attachedto a terminal carbon atom of (CH₂)_(m), the method comprising:reacting aterminal moiety-containing compound with a suitable base, solvent andsubstituted olefin to obtain an intermediate product, the intermediateproduct having a composite structure of the terminal moiety-containingcompound and substituted olefin; either: A) converting the intermediateproduct to a terminal moiety-containing epoxide in a reaction withorganic peracid; or B) first, converting the intermediate product to acorresponding diol in a reaction with a suitable oxidizing agent;second, reacting the corresponding diol using a halogenating agent inthe presence of an organic acid to obtain a haloester; and third,converting the haloester to a terminal moiety-containing epoxide byreaction of the haloester and a basic ester-hydrolyzing reagent; andreacting the terminal moiety-containing epoxide with a substituted orunsubstituted amine to obtain the compound.
 2. The method of claim 1,wherein the substituted olefin has at least one additional functionalmoiety which may be displaced in a chemical reaction with the terminalmoiety-containing compound.
 3. The method of claim 2, wherein the firstreacting step further comprises:chemically converting the terminalmoiety-containing compound to a terminal moiety-containing anion.
 4. Themethod of claim 3, wherein the terminal moiety-containing anion isreacted with the substituted olefin, displacing the at least onefunctional moiety to obtain the intermediate product.
 5. The method ofclaim 1, wherein the suitable base is selected from the group consistingof sodium hydride, sodium amide, sodium alkoxide, lithium hydride,potassium hydride, lithium amide, sodium amide and potassium amide. 6.The method of claim 1, wherein the solvent is selected from the groupconsisting of dimethylsulfoxide, dimethylformamide, or an alcohol. 7.The method of claim 1, wherein the substituted olefin comprises a chainstructure corresponding to (CH₂)_(m).
 8. The method of claim 7, whereinthe substituted olefin is an ω-substituted olefin.
 9. The method ofclaim 1, wherein the substituted olefin is a halo-substituted olefin.10. The method of claim 1, wherein the organic peracid is selected fromthe group consisting of 3-chloroperoxybenzoic acid, peracetic acid andtrifluoroperacetic acid.
 11. The method of claim 1, wherein theoxidizing agent is osmium tetroxide.
 12. The method of claim 1, whereinconverting the intermediate product to a corresponding diol furthercomprises reacting the intermediate product with a catalytic amount ofthe oxidizing agent in the presence of a regenerating agent.
 13. Themethod of claim 12, wherein the regenerating agent is4-methylmorpholine-N-oxide or trimethylamine-N-oxide.
 14. The method ofclaim 1, wherein the halogenating agent is selected from the groupconsisting of hydrogen bromide and hydrogen chloride.
 15. The method ofclaim 1, wherein the organic acid is selected from the group consistingof acetic acid and propionic acid.
 16. The method of claim 1, whereinthe ester-hydrolyzing agent is selected from the group consisting of ametal alkoxide and metal hydroxide.
 17. The method of claim 16, whereinthe metal alkoxide is selected from the group consisting of sodiummethoxide, ethoxide, isopropoxide and pentoxide.
 18. The method of claim16, wherein the metal hydroxide is sodium hydroxide.
 19. The method ofclaim 1, wherein reacting the terminal moiety-containing epoxide with asubstituted or unsubstituted amine comprises either:A) heating theterminal moiety-containing epoxide in the presence of an amine, havingamine substituents present in the compound; or B) reacting the terminalmoiety-containing epoxide with an amine, having substituents present inthe compound, with a reaction activator in a solvent.
 20. The method ofclaim 19, wherein the reaction activator is lithium perchlorate.
 21. Themethod of claim 19, wherein the solvent is selected from the groupconsisting of dimethylsulfoxide, dimethylformamide, or an alcohol. 22.The method of claim 19, wherein the substituted or unsubstituted aminehas a molecular structure corresponding to ##STR109##